CN115261496A - Method for detecting salmonella, application and primer thereof - Google Patents
Method for detecting salmonella, application and primer thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting salmonella, application and primers thereof. The method comprises the following steps: identifying a sequence rich in AT base in a salmonella genome sequence as a target sequence; designing specific primers aiming at a target sequence automatically in a high-throughput manner; designing a specific Tm value calculation formula to calculate the reaction denaturation and annealing temperatures, and setting the nucleic acid amplification reaction conditions according to the reaction denaturation and annealing temperatures; the nucleic acid amplification reaction is performed under conditions in which the template is locally melted. The target-oriented traditional nucleic acid amplification method generally needs to avoid sequences rich in AT bases, so that the candidate target area is greatly widened; by means of specific examination based on massive genome data and accurate calculation of Tm value of AT-rich area, the local melting of double-stranded DNA is realized, and the possibility of non-specific amplification in nucleic acid amplification technology is greatly reduced. The method effectively expands the application range of the traditional nucleic acid amplification technology and obviously improves the reaction performance.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting salmonella, application and primers thereof.
Background
Nucleic Acid Amplification Technology (NAAT) is a generic name of a class of molecular biology technologies, which can achieve rapid and specific amplification of trace nucleic acids by reacting primers, DNA polymerase and other reagents at a specific temperature, and can be widely applied to disease diagnosis, pathogenic microorganism detection, food safety detection, animal and plant quarantine, and various applications related to molecular cloning, such as sequencing, gene cloning, gene manipulation, allele analysis, mutation detection, and the like. The Polymerase Chain Reaction (PCR) is the earliest nucleic acid amplification technology, which simulates the process of DNA replication in vivo, and realizes exponential amplification of DNA fragments through the complementation of a pair of specific oligonucleotide primers and the DNA fragments to be amplified, and through several cycles of denaturation, annealing and extension. The PCR technology was invented in 1985 by Mullis of Cetus company, and then Saiki et al introduced thermostable DNA polymerase into the PCR reaction system, which solves the problem of continuous manual addition of polymerase caused by inactivation of DNA polymerase in the thermal denaturation cycle process in the early PCR technology, greatly improves the nucleic acid amplification efficiency and enables the technology to be automated, and since then, the nucleic acid amplification technology represented by PCR is widely popularized and applied, and the development of molecular biology is greatly promoted.
It is well known that existing nucleic acid amplification techniques generally avoid GC-or AT-rich regions because these regions tend to form hairpin secondary structures by self-complementary pairing, thereby hindering primer binding to the template; even if the primer is capable of binding to the template only marginally, it is susceptible to extension of the template strand by the DNA polymerase, resulting in "katton" of the DNA polymerase as it is amplified along the template and interfering with DNA synthesis. Therefore, in order to improve the success rate of the reaction, the conventional nucleic acid amplification technology has a selective preference for the base composition of the amplification region, the primer design is often concentrated in the region with a GC content of 40% to 60%, 45% to 55% is most suitable, and the different amplification technologies are slightly different, such as the primer design of PCR suggests that the G + C content is 40% to 60% (see the fourth edition of molecular cloning experimental guidelines, table 7-1 primer design), and the LAMP primer design suggests that the G + C content is 40% to 65% (see a Guide to LAMP primer design (primerexplorer v 3)).
However, in nature, the nucleotide composition of the nucleic acid of organisms is very different, for example, the AT nucleotide content of plasmodium genome is about 82%, and the GC content of the genome of 2670 strains of bacteria and archaea is found to vary from 14% to 75% (Zhou Hui Qi, 2014). From the detection perspective, the base composition selection preference of the existing nucleic acid amplification technology ignores a large number of potential target sequences, and amplifies the difficulty of successfully detecting a target object; from a genetic manipulation perspective, this selective preference makes it difficult for a significant number of target sequences to be successfully amplified, thereby making further molecular manipulation difficult. If proper primers can be designed aiming AT the unbalanced domains of GC base content and AT base content avoided by the traditional nucleic acid amplification method, the formation of a secondary structure is avoided, and meanwhile, proper reaction conditions are set, so that a nucleic acid amplification experiment is smoothly carried out, the success rate of detecting a target object is obviously increased, and more sequences can be amplified to conveniently implement downstream molecular operation.
The method is characterized in that a nucleic acid amplification reaction is carried out aiming AT a region with non-equilibrium GC base content and AT base content, and the key step is a primer design link. In view of the above analysis, the species-specific genes (or sequences) published in the field are usually from a region with a GC content of 40-60% or even 45-55%, and thus cannot be applied to primer design for regions with non-equilibrium base content of GC and AT. In addition, the continuous GC-rich or AT-rich region in such sequences has a weak diversity of base composition compared to other regions, which brings additional difficulties in designing specific primers. Therefore, a primer design algorithm process with high throughput, automation and high efficiency aiming AT the base content non-equilibrium sequences of GC and AT needs to be provided.
In addition to the selection of the amplification region and the design of the primers, the calculation of Tm values of the amplification region and the primers is also an important influence factor on the smooth completion of the amplification process. The Tm values of the amplified region and the primers are usually calculated according to the nearest neighbor binary model, but the specific calculation formulas used by different researchers and primer manufacturers are different, for example, the Tm calculation formula of the primers proposed in the fourth edition of the molecular cloning experimental instruction manual is Tm =4 (G, C) +2 (A, T), and the Tm calculation formula of the primers proposed by TaKaRa is Tm =4 (G, C) +2 (A, T) +32-2 (total base); the Tm formula of the primers of the organism is Tm (0.05M Na)+) =59.94+1 × (percentage content of GC) - (675/primer sequence length). In the region with non-equilibrium base content of GC and AT, how to design a model to calculate the Tm value of the primer becomes an important factor for ensuring the success of the experiment.
In summary, there is a need in the art to develop a nucleic acid amplification technology for regions that are difficult to be involved in the conventional nucleic acid amplification technology, which can automatically design specific primers with high throughput, calculate Tm values thereof, set appropriate reaction conditions such as denaturation temperature and annealing temperature, expand the application range of the nucleic acid amplification technology, improve the amplification specificity, and satisfy the requirements of nucleic acid detection, molecular genetics research and other aspects.
Disclosure of Invention
The invention provides an innovative nucleic acid amplification method and is applied to detecting salmonella. The method takes an AT-rich nucleic acid sequence as an amplification target, and carries out target sequence identification, primer design, denaturation temperature and annealing temperature calculation, nucleic acid amplification of template local melting and the like. Firstly, an automatic primer design flow is adopted, abundant genome sequence information in public data resources is fully utilized, and a specific primer pair aiming AT an AT-rich target sequence is designed in a high-throughput manner. Secondly, fitting a Tm value calculation formula conforming to the characteristics of the AT-rich sequence, and calculating the denaturation temperature and the annealing temperature according to the theoretical amplification product sequence, the sequence length of the primer, the base composition and other factors; considering that the denaturation temperature of the AT-rich nucleic acid sequence is significantly lower than that of the sequences with GC content of 40-60% and high GC content, the calculated Tm value of the theoretical amplification product sequence is used as the lowest denaturation temperature, and AT the lowest denaturation temperature, the AT-rich region of the double-stranded DNA can be locally melted without adding any chemical denaturant, so that the nucleic acid amplification reaction can be started.
On one hand, massive genome data in public data resources are introduced in a primer design link to carry out sequence specificity investigation; on the other hand, in the denaturation temperature setting link, the method is different from the traditional nucleic acid amplification method that all double-stranded structures are opened AT the denaturation temperature of 93-95 ℃, but only AT-rich sequences are opened by reducing the denaturation temperature, so that the possibility of non-specific amplification of non-target regions is greatly reduced AT the source. The design strategies of the two aspects remarkably reduce the possibility of non-specific amplification common in nucleic acid amplification reaction, and a nucleic acid amplification method aiming AT AT-rich sequences is formed on the basis of the non-specific amplification strategy. The primer design step of the method is realized by adopting C language and Perl language programming, the method has the characteristics of high throughput and automation, the whole amplification method has strong specificity and high success rate, the application range of the traditional nucleic acid amplification technology is effectively expanded, and the reaction performance is obviously improved.
In the present invention, the nucleic acid amplification method comprises identifying a sequence rich in AT base in a nucleic acid sequence to be amplified as a target sequence; designing specific primers aiming at a target sequence automatically in a high-throughput manner; designing a specific Tm value calculation formula to calculate the reaction denaturation and annealing temperatures, and setting the nucleic acid amplification reaction conditions according to the reaction denaturation and annealing temperatures; the nucleic acid amplification reaction is performed under conditions in which the template is locally melted.
The method specifically comprises the following steps:
(1) Screening a target sequence rich in AT basic groups in a nucleic acid sequence to be amplified;
(2) Designing primers with universality and specificity aiming at the target sequence automatically and in high throughput;
(3) Calculating the reaction denaturation temperature and the annealing temperature by a specific formula, and setting the reaction conditions of nucleic acid amplification; calculating the inverse dependent temperature and the annealing temperature based on the percentage content of GC, the sequence length of a primer and the sequence length of a theoretical amplification product of a primer pair, and setting the reaction conditions of nucleic acid amplification;
(4) And carrying out nucleic acid amplification reaction under the condition of local melting of the template to obtain an amplification product.
In the invention, the method for detecting the salmonella specifically comprises the following steps:
(1) Screening a target sequence rich in AT base in a salmonella genome sequence;
(2) Designing a primer with both universality and specificity aiming at the target sequence;
(3) Calculating the inverse strain temperature and the annealing temperature based on the percentage content of GC, the sequence length of the primer and the sequence length of a theoretical amplification product of the primer pair, and setting the reaction conditions of nucleic acid amplification;
(4) And carrying out nucleic acid amplification reaction under the condition of local melting of the template to obtain an amplification product.
In the invention, the primer pair is as follows:
and (3) primer pair A:
Sal-F1：5’-TGGGTTGAAATAGCCCATTA-3’(SEQ ID NO.1)
Sal-R1：5’-GACGTGACACACTTCGTTTT-3’(SEQ ID NO.2)。
in step (1) of the present invention, for the nucleic acid sequence to be amplified, a window with a sliding width of 1000bp starting from the first base is used, and the step length is 5-100 bp. Calculating the AT base content of the sequence contained in the position of each window, and reserving the region with the AT base content of the sequence more than 60 percent as the target sequence. Preferably, the target sequence is retained in a region having a base content of 60 to 80% of the AT sequence.
In the step (1), the salmonella genome sequence is slid by a window with the width of 1000bp from the first base, and the step length is 5-100 bp. Calculating the AT base content of the sequence contained in the position of each window, and reserving the region with the AT base content of the sequence more than 60 percent as the target sequence. Preferably, the region with the base content of AT sequence of 60-80% is reserved as the target sequence.
In step (2) of the present invention, the method for designing the primer comprises: (2.1) designing a single primer aiming at a target sequence to obtain a candidate primer; (2.2) judging the physicochemical properties of the candidate primers, and screening single primers meeting the conditions; (2.3) combining the single primers obtained by screening in the step (2.2) into a primer pair; (2.4) judging the universality and the specificity of the primer pair; and (2.5) outputting a primer pair meeting the conditions to obtain the specific primer.
In the invention, any programming language capable of realizing high-throughput primer design can be adopted, such as C, perl and other programming languages with strong operability and higher speed.
In the step (2.1), a candidate primer is designed aiming at a target sequence, and the candidate primer needs to satisfy the following conditions: a) The length of the primer sequence is between 20bp and 36 bp; b) The AT base content is 55-80%, c) the continuous GC number is less than or equal to 5; and simultaneously recording the position information and the positive and negative chain information of the candidate primers matched on the target sequence.
Wherein, preferably, the AT base content is 60% -75%.
Wherein the "consecutive GC number" refers to the number of consecutive bases G or consecutive bases C in the primer sequence, for example, the number of consecutive GCs in the primer sequence AAGGGGGTTCCAGGCATTA (SEQ ID NO. 15) is 5/2/2.
In the step (2.2), the single primer meeting the condition (2.1) is subjected to physicochemical property judgment, including but not limited to physicochemical properties such as 3 'end stability, 5' end stability and/or secondary structure stability, and the single primer meeting the requirement is reserved.
Wherein, the 'single primer meeting the requirement' refers to the single primer with 3 'end stability, 5' end stability and secondary structure stability.
In the step (2.3), the single primers obtained by screening in the step (2.2) are combined into a primer pair according to the position information and the positive and negative chain information matched with the target sequence; the length of the theoretical amplification product sequence of the primer pair should be between 200bp and 600 bp. Calculating Tm value of single primer according to the formula 0.466X (GC percentage content) 100+66.04- (450/primer sequence length); and screening the primer pairs under the conditions that the Tm difference value of the primer pairs is less than or equal to 3 ℃ and the primers cannot interact with each other to obtain candidate primer pairs. Wherein, the percentage content of GC refers to the percentage of the number of bases G and C in the primer to the total number of bases of the primer; the primer sequence length refers to the number of bases of the primer.
In the step (2.4), the universality and specificity of each primer in the candidate primer pair obtained in the step (3) are judged. Wherein the determination of commonality refers to checking whether the primer pairs are exactly matched to all target sequences (e.g., multiple strain genomes of Salmonella);
the specificity determination refers to checking whether a single primer cannot be specifically matched with non-target sequences except for all target sequences, the non-target sequences refer to nucleic acid sequences except for the nucleic acid sequence to be amplified, and the specific matching refers to matching with no more than 2 mismatches.
The candidate primers determined by the generality and specificity can proceed to the next step.
And (2.5) outputting a primer pair meeting the conditions to obtain the primer pair for the nucleic acid amplification reaction.
In step (3) of the present invention, the calculation formula of the reaction denaturation temperature is 0.357X (GC percentage content) × 100+70.582- (990/theoretical amplification product sequence length of primer pair), which is recorded as Tma; wherein, the percentage content of GC is the percentage of the number of bases G and C in the theoretical amplification product sequence of the primer pair to the total number of bases of the theoretical amplification product sequence of the primer pair.
In the step (3) of the present invention, the reaction annealing temperature is an average value of Tm values of the two primers in the step (2.3) and is denoted as Tmb. Namely, calculating the Tm value of a single primer according to the formula 0.466X (GC percentage content) X100 +66.04- (450/primer sequence length), and taking the average value of the Tm values as the reaction annealing temperature, and marking as Tmb; wherein, the percentage content of GC refers to the percentage of the number of bases G and C in the primer to the total number of bases of the primer.
In the step (3) of the present invention, the nucleic acid amplification reaction conditions include: the reaction was carried out at the denaturation temperature for 5 seconds, at the annealing temperature for 5 seconds and at the elongation temperature for 20 seconds, and the above procedure was repeated 30 to 40 times. Wherein the denaturation temperature can be adjusted within the range of Tma +/-5 ℃ according to the requirement; the annealing temperature can be adjusted between Tmb plus or minus 2 ℃ according to the requirement; the extension temperature was 72 ℃.
The invention also provides application of the method in nucleic acid amplification of the target nucleic acid sequence rich in the AT region.
The invention also provides a primer pair obtained by the design method.
In the invention, the primer pair is as follows:
primer pair A:
Sal-F1：5’-TGGGTTGAAATAGCCCATTA-3’(SEQ ID NO.1)
Sal-R1：5’-GACGTGACACACTTCGTTTT-3’(SEQ ID NO.2)
the invention also provides the use of a method as described above or a primer pair as described above for the amplification and/or detection of a salmonella-related gene or region.
The invention also provides application of the primer pair in preparation of products for amplifying and/or detecting related genes or regions of salmonella.
The invention also provides application of the method in simultaneously detecting any two or more combinations of salmonella, staphylococcus aureus, cronobacter sakazakii and yersinia enterocolitica.
Specifically, the invention also provides the application of the method in simultaneously detecting any one of the salmonella and staphylococcus aureus, sakazakii Cronobacter sakazakii and Yersinia enterocolitica and the combination of the above.
The invention provides a reagent for diagnosing salmonella and application thereof in detecting the salmonella, wherein the diagnostic reagent comprises the primer pair.
The invention also provides a diagnostic reagent and an application thereof in detecting salmonella, wherein the diagnostic reagent comprises the primer pair.
The present invention also provides a system for detecting salmonella, the system comprising:
the screening module is used for screening a target sequence which is rich in AT base in the salmonella genome sequence;
a primer design module for designing a primer having both versatility and specificity for the target sequence;
the calculation module is used for calculating the inverse strain temperature and the annealing temperature based on the percentage content of GC, the length of the primer sequence and the length of the theoretical amplification product sequence of the primer pair, and setting the reaction conditions of nucleic acid amplification;
and the amplification module is used for carrying out nucleic acid amplification reaction under the condition of local melting of the template to obtain an amplification product.
The invention has the advantages that the target traditional nucleic acid amplification method usually needs to avoid sequences rich in AT bases, thereby greatly expanding candidate target regions; by specific investigation based on massive genome data and accurate calculation of Tm value of AT-rich region, double-stranded DNA is locally unzipped, and the possibility of non-specific amplification commonly existing in nucleic acid amplification technology is greatly reduced. The method effectively expands the application range of the traditional nucleic acid amplification technology, obviously improves the reaction performance in the aspects of primer design flux, reaction specificity and the like, and can meet the requirements of various aspects such as nucleic acid detection, molecular genetics research and the like.
Drawings
FIG. 1 is a graph showing the amplification curve of real-time fluorescent quantitative PCR based on the nucleic acid amplification reaction of the Salmonella (Salmonella) primer pair Sal-F1/R1 of the present invention at an appropriate temperature.
FIG. 2 is a melting curve of real-time fluorescent quantitative PCR based on the nucleic acid amplification reaction of the Salmonella (Salmonella) primer pair Sal-F1/R1 of the present invention at an appropriate temperature.
FIG. 3 is an agarose gel electrophoresis at a suitable temperature based on the nucleic acid amplification reaction of the Salmonella (Salmonella) primer pair Sal-F1/R1 of the present invention.
FIG. 4 shows the sensitivity of real-time fluorescent quantitative PCR based on the Salmonella (Salmonella) primer pair Sal-F1/R1 of the present invention to denaturation at 80 ℃ with 4 Salmonella as templates.
FIG. 5 shows the sensitivity of real-time fluorescent quantitative PCR based on the Salmonella (Salmonella) primer pair Sal-F1/R1 of the present invention against denaturation at 81 ℃ of 4 Salmonella as templates.
FIG. 6-1 shows the sensitivity of the primer pair Sal-F1/R1 based on Salmonella (Salmonella) of the present invention to fluorescent dye method detection in PCR with 4 types of Salmonella as templates denatured at 80 ℃.
FIG. 6-2 shows the sensitivity of the primer pair Sal-F1/R1 based on Salmonella (Salmonella) of the present invention to PCR denaturation of 4 species of Salmonella as templates at 81 ℃ by the fluorescent dye method.
FIG. 7-1 shows the sensitivity of PCR based on the Salmonella (Salmonella) primer pair Sal-F1/R1 of the present invention for denaturation of 4 species of Salmonella as templates at 80 ℃ for detection by agarose gel electrophoresis.
FIG. 7-2 shows the sensitivity of PCR based on the Salmonella (Salmonella) primer pair Sal-F1/R1 of the present invention for denaturation of 4 species of Salmonella as templates at 81 ℃ for detection by agarose gel electrophoresis.
FIG. 8 shows the results of staining with a fluorescent dye for specific detection of nucleic acid amplification at a denaturation temperature of 81 ℃ based on the nucleic acid amplification reaction of the Salmonella (Salmonella) primer pair Sal-F1/R1 of the present invention.
FIG. 9 shows the results of dye development based on the detection of nucleic acid amplification specificity at different denaturation temperatures of Cronobacters sakazakii (Cronobacters sazakii) according to the present invention.
FIG. 10 shows the results of electrophoresis of amplification products for detection of nucleic acid amplification specificity at a denaturation temperature of 94 ℃ based on Cronobacter sakazakii (Cronobacter sazakii) of the present invention.
FIG. 11 shows the results of electrophoresis of amplification products based on the detection of nucleic acid amplification specificity at a denaturation temperature of 82 ℃ of Cronobacters sakazakii (Cronobacters sazakii) according to the present invention.
FIG. 12 shows the results of the fluorescent dye staining of the nucleic acid amplification reaction of the present invention against four bacteria, salmonella enteritidis subspecies, staphylococcus aureus subspecies, yersinia enterocolitica, and Cronobacter sakazakii.
FIG. 13 shows the results of agarose gel electrophoresis of the nucleic acid amplification reactions of the present invention against four species of bacteria, salmonella enteritidis subspecies, staphylococcus aureus subspecies, yersinia enterocolitica, and Cronobacter sakazakii.
FIG. 14 shows the results of the color development of SYBRGreen I dye in the specific detection of the nucleic acid amplification reaction of the present invention against four bacteria, salmonella enterica, staphylococcus aureus, yersinia enterocolitica, and Cronobacter sakazakii, at a denaturation temperature of 81 ℃.
FIG. 15 shows the result of agarose gel electrophoresis of the nucleic acid amplification reaction of two bacteria, salmonella enteritidis subspecies and Staphylococcus aureus subspecies, according to the present invention.
FIG. 16 shows the result of agarose gel electrophoresis of the nucleic acid amplification reaction of two bacteria, salmonella enteritidis subspecies and Yersinia enterocolitica, according to the present invention.
FIG. 17 shows the result of agarose gel electrophoresis of nucleic acid amplification reactions of three bacteria, salmonella enteritidis subspecies, staphylococcus aureus subspecies and Yersinia enterocolitica, according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
The present invention also provides a system for detecting salmonella, the system comprising:
the screening module is used for screening a target sequence which is rich in AT base in the salmonella genome sequence;
a primer design module for designing a primer having both versatility and specificity for the target sequence;
the calculation module is used for calculating the inverse strain temperature and the annealing temperature based on the percentage content of GC, the length of the primer sequence and the length of the theoretical amplification product sequence of the primer pair, and setting the reaction conditions of nucleic acid amplification;
and the amplification module is used for carrying out nucleic acid amplification reaction under the condition of local melting of the template to obtain an amplification product.
Example 1 detection of Salmonella (Salmonella)
The method comprises the steps of screening sequences rich in AT aiming AT Salmonella (Salmonella) genomes, designing specific primers, setting reaction conditions according to the calculated primer Tm and the target sequence Tm for nucleic acid amplification, determining whether a target sequence exists in a sample to be detected or not by judging whether a reaction result is positive or not, and further determining whether Salmonella exists in the sample to be detected or not. The method comprises the following specific steps:
(1) Screening of target sequences rich in AT bases:
2896 whole genome sequences were used for bacteria, archaea and virus data with complete genome sequences downloaded from the FTP at NCBI at 8/5 in 2019. Set a, which contains all salmonella genomic sequences; set B was set, which contained all non-salmonella genomic sequences. The salmonella genome sequence is used as a reference genome, a window with the width of 1000bp slides from the first base of the genome, and the step length is 50bp; calculating the base content of the sequence AT once before each sliding, and reserving a region with the base content of the sequence AT more than 60% as a candidate target sequence. The above process is implemented using perl scripts.
(2) Design of specific primers:
according to the characteristics of the PCR primer and the requirements of the invention, the characteristic parameters of the primer, such as the AT base content of 55-75%, the 3 'end stability delta G <4, the 5' end stability delta G <3, the primer sequence length (20-36 bp) and the like, are set, meanwhile, the conditions that a single primer cannot generate a hairpin structure and cannot interact with the single primer are set, and the single primer meeting the set conditions is calculated by taking the candidate target sequence in the step (1) as a candidate sequence for designing the single primer. The Tm value of a single primer is calculated by the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length), and the position of each primer on the target sequence, the plus and minus chain information (i.e., whether derived from the plus chain or the minus chain), and the length of the primer are recorded.
And (3) carrying out primer pairing according to the position information of the single primer, and reserving a primer pair which simultaneously meets the conditions that the Tm difference of the primer pair is less than 3 ℃ and the amplification area of the primer pair is between 200 and 600bp as a candidate primer pair.
And (3) using alignment software Bowtie to perform sequence alignment on each primer in the candidate primer pair designed in the last step with the target genome sequence in the set A and the non-target genome sequence in the set B respectively. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in the set A, the parameter setting of '-a-n 0' is used, namely the single primer is required to be completely matched with the target sequence; to ensure primer specificity, when the single primer is aligned to the non-target sequences in pool B, a parameter setting of "-a-n 3" is used, i.e., the single primer is allowed to have no more than 3 mismatches to the non-target sequences. The system outputs primer pairs satisfying the condition, in which the number of primer pairs is preset, and in this embodiment, the number of primer pairs is preset to 20. The high-throughput and automatic primer design process is realized by adopting C and Perl scripts.
After the program is operated, 20 PCR reaction primer pairs are designed in the AT enrichment region, and one primer group is randomly selected for validity verification. The sequences of the primer pairs are as follows:
Sal-F1：5’-TGGGTTGAAATAGCCCATTA-3’(SEQ ID NO.1)
Sal-R1：5’-GACGTGACACACTTCGTTTT-3’(SEQ ID NO.2)
the AT base content of the primer is respectively 60 percent and 55 percent, and the percentage content of the theoretical amplification product sequence AT of the primer pair is 74.43 percent.
Calculation of denaturation temperature and annealing temperature of PCR reaction:
tm =62.18 ℃ for primer Sal-F, tm =64.51 ℃ for Sal-R, calculated using the formula 0.466X (GC percentage) X100 +66.04- (450/primer sequence length), the primer pair average Tm value being 63.35 ℃; the denaturation temperature of the amplified region was calculated to be 76.90 ℃ using the formula 0.357X (GC percentage) X100 +70.582- (990/amplification product sequence length).
(4) Nucleic acid amplification reaction and result detection:
the nucleic acid amplification reaction system configuration is shown in table 1 below. Determining an applicable denaturation temperature and a dissolution curve by using a real-time fluorescence quantitative PCR instrument according to the calculation result; the specificity of the primers for different detection objects at the applicable denaturation temperature is tested; the limit of detection at the applicable denaturation temperature was tested. Denaturation for 5 seconds, annealing at 60 ℃ for 5 seconds, and extension at 72 ℃ for 20 seconds, and it is recommended to repeat the above process 35 times. The list of specific test subjects is shown in Table 2.
After the reaction is finished by using a common gradient PCR instrument, the amplification result can be judged by two modes, namely, firstly, SYBRGreen I dye with the final concentration of 25x is added, whether the amplification result is positive or not is judged by color, namely whether a target sequence exists in a sample to be detected or not is judged; secondly, agarose gel electrophoresis is carried out on the amplification product, and whether the amplification result is positive or not is judged according to the electrophoresis strip, namely whether the target sequence exists in the sample to be detected or not is judged.
TABLE 1 Salmonella (Salmonella) nucleic acid amplification reaction System
| System of | Volume (μ l) | Final concentration |
| R300 MIX(TaKaRaTaqTM HS Perfect Mix) | 12.5 | / |
| Sal-F/Sal-R(10μM) | 1.25+1.25 | 0.5μM |
| 100％DMSO | 0.5 | 2 |
| DNA template | ||
| 0/1 | / | |
| ddH2O | Up to 25 | / |
TABLE 2 List of objects for specific detection of Salmonella (Salmonella) nucleic acid amplification reaction
Wherein 1 to 9 are respectively staphylococcus aureus, staphylococcus aureus subspecies, staphylococcus epidermidis, rhodococcus equi, bacillus cereus, bacillus mycoides, listeria monocytogenes, listeria inonotus and listeria israeli, 14 to 30 are respectively shigella dysenteriae, shigella baumannii, shigella flexneri, escherichia coli (containing clostridium botulinum type A gene), pathogenic escherichia coli, enterotoxigenic escherichia coli, escherichia coli enterotoxigenic, escherichia coli enterohemorrhagic, clostridium sakazakii, shigella enterocolitica, yersinia pseudotuberculosis, vibrio vulnificus, vibrio parahaemolyticus, vibrio francisci, cholera cholerae and shigella sonnei, N: negative control; 10-13 are salmonella enteritidis subspecies, salmonella enteritidis, salmonella typhimurium and salmonella paratyphi B respectively.
FIGS. 1 to 3 show the amplification curve, the dissolution curve and the agarose gel electrophoresis result of the real-time fluorescence quantitative PCR of the nucleic acid amplification reaction of Salmonella (Salmonella) in the invention at a suitable temperature, wherein "80 ℃", "81 ℃" indicates the amplification result of the positive template at the denaturation temperature, "NTC" or "N" indicates the corresponding amplification result of the negative template, and "M" indicates Marker DL2000. FIG. 1 shows that amplification conditions of positive templates and negative templates of Salmonella enteritidis subspecies, salmonella enteritidis, salmonella typhimurium, and Salmonella paratyphi B are expected. FIG. 2 shows that the solubility curves of Salmonella enteritidis subspecies, salmonella enteritidis, salmonella typhimurium, and Salmonella paratyphi B are all single peaks and have no non-specific reaction. FIG. 3 shows the results of the amplification electrophoresis identification with 81 ℃ and 82 ℃ denaturation on the left and right sides of M ", and if a single band is present at 352bp after the electrophoresis of the amplification product, the result is positive; if the amplified product has no band after electrophoresis, the amplified product is negative.
FIGS. 4 to 7 show 3 detection limit detection methods of the present invention for nucleic acid amplification reaction of Salmonella (Salmonella) at denaturation temperatures of 80 ℃ and 81 ℃. FIG. 4/5 and Table 3, 10ng to 10fg was diluted 10-fold; in the range of 10ng to 100pg, amplification curves differed more than 3 cycles per 10-fold difference, and were less linear than 10 pg. Therefore, the standard curve is made above 10pg, and quantitative determination can be carried out; FIG. 6 shows the sensitivity of the addition of SYBRGreen I dye at a final concentration of 25X, with 10ng to 10fg dilution, and positive results in the range of 10ng to 100pg (FIGS. 6-1 and 6-2 show the sensitivity of the primer pair Sal-F1/R1 based on Salmonella (Salmonella) of the present invention, respectively, in PCR with denaturation at 80 ℃ and 81 ℃ using 4 Salmonella as templates, by fluorescent dye method). FIG. 7 shows the results of agarose gel electrophoresis of the amplification products, showing a single band at 352bp, which was judged to be positive (FIGS. 7-1 and 7-2 show the sensitivity of the primer pair Sal-F1/R1 based on Salmonella (Salmonella) of the present invention to PCR denaturation of 4 species of Salmonella at 80 ℃ and 81 ℃ by agarose gel electrophoresis, respectively).
TABLE 3 FIG. 4/5 summary of real-time fluorescent quantitative PCR sensitivity
FIG. 8 shows the results of the color development of SYBRGreen I dye in the specific detection of the nucleic acid amplification reaction of the present invention against Salmonella (Salmonella) at a denaturation temperature of 81 ℃. If the amplification product is bright green, the amplification product is positive; if the amplification product is orange, the amplification product is negative. In fig. 8, the negative control (N) appears orange, a negative result, as expected; the positive control (P) is green, and is a positive result, which is in line with the expectation. No. 10-13 salmonella enteritidis subspecies, salmonella enteritidis, salmonella typhimurium and salmonella paratyphi B present bright green, are positive results and accord with expectations, and meanwhile, the detection results of other bacterial genome DNA templates are negative, as shown in the No. 1-9 and No. 14-30 tubes. All results were as expected, suggesting that no or even a slight amount of non-specific amplification occurred at the denaturation temperature of 81 ℃ but not enough to affect the dye-based outcome determination. Example 2 detection of Cronobacter sakazakii (Cronobacters sazakii)
The genome targeted by the designed primer is changed into a genome of Cronobacter sakazakii (Cronobacters sakazakii), an AT-rich sequence is screened, a specific primer is designed, reaction conditions are set according to the calculated primer Tm and the target sequence Tm for nucleic acid amplification, whether the target sequence exists in a sample to be detected is determined by judging whether the reaction result is positive, and further whether the Cronobacter sakazakii exists in the sample to be detected is determined. The method comprises the following specific steps:
(1) Screening of target sequences rich in AT bases:
2896 whole genome sequences were used for bacteria, archaea and virus data with complete genome sequences downloaded from the FTP at NCBI at 8/5 in 2019. Set a, which comprises all cronobacter sakazakii genomic sequences; set B, which comprises all sakazakii non-sakazakii genomic sequences, was set. Taking a cronobacter sakazakii genome sequence with the GI number of 156932229 as a reference genome, sliding a window with the width of 1000bp from the first base of the genome, and setting the step length as 50bp; calculating the base content of the sequence AT once before each sliding, and reserving a region with the base content of the sequence AT more than 60% as a candidate target sequence. The above process is implemented using perl scripts.
(2) Design of specific primers:
according to the characteristics of the PCR primer and the requirements of the invention, the characteristic parameters of the primer, such as the AT base content of 55-75%, the 3 'end stability delta G <4, the 5' end stability delta G <3, the primer sequence length (20-36 bp) and the like, are set, meanwhile, the conditions that a single primer cannot generate a hairpin structure and cannot interact with the single primer are set, and the single primer meeting the set conditions is calculated by taking the candidate target sequence in the step (1) as a candidate sequence for designing the single primer. The Tm value of a single primer is calculated by the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length), and the position of each primer on the target sequence, the plus and minus chain information (i.e., whether derived from the plus chain or the minus chain), and the length of the primer are recorded.
And (3) carrying out primer pairing according to the position information of the single primer, and reserving a primer pair which simultaneously meets the two conditions that the Tm difference of the primer pair is less than 3 ℃ and the amplification area of the primer pair is 200-600 bp as a candidate primer pair.
And (3) using alignment software Bowtie to perform sequence alignment on each primer in the candidate primer pair designed in the last step with the target genome sequence in the set A and the non-target genome sequence in the set B respectively. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in the set A, the parameter setting of '-a-n 0' is used, namely the single primer is required to be completely matched with the target sequence; to ensure primer specificity, when the single primer is aligned to the non-target sequences in pool B, a parameter setting of "-a-n 3" is used, i.e., the single primer is allowed to have no more than 3 mismatches to the non-target sequences. The system outputs primer pairs satisfying the condition, in which the number of primer pairs is preset, and in this embodiment, the number of primer pairs is preset to 20. The high-throughput and automatic primer design process is realized by adopting C and Perl scripts.
After the program is operated, 20 PCR reaction primer pairs are designed in the AT enrichment region, and one primer group is randomly selected for validity verification. The sequences of the primer pairs are as follows:
Cro-F：5’-CGCCATAACTGCATAATCAT-3’(SEQ ID NO.3)
Cro-R：5’-ATAACGAGTTACCGTGCAGA-3’(SEQ ID NO.4)
the AT base content of the primer is respectively 60 percent and 55 percent, and the percentage content of the theoretical amplification product sequence AT of the primer pair is 69 percent.
(3) Calculation of denaturation temperature and annealing temperature for PCR reaction:
tm =62.2 ℃ for primer Cro-F, tm =64.5 ℃ for Cro-R, calculated using the formula 0.466X (GC percentage) X100 +66.04- (450/primer sequence length), the primer pair average Tm value is 63.4 ℃; the denaturation temperature of the amplification region was calculated to be 78.3 ℃ using the formula 0.357X (percent GC content). Times.100 +70.582- (990/length of amplification product sequence).
(4) Nucleic acid amplification reaction and result detection:
the nucleic acid amplification reaction system configuration is shown in Table 4 below. According to the calculation result, 3 groups of experiments are set, the specificity of the primers for different detection objects at the denaturation temperature of 94 ℃/90 ℃/82 ℃ is respectively tested, the nucleic acid amplification reaction conditions are 94 ℃/90 ℃/82 ℃ denaturation for 5 seconds, 63 ℃ annealing for 5 seconds and 72 ℃ extension for 20 seconds, and the processes are repeated for 35 times. The list of specific test subjects is shown in Table 5.
After the reaction is finished, judging the amplification result by two modes, namely adding SYBRGreen I dye with the final concentration of 25x, and judging whether the amplification result is positive or not by color, namely whether a target sequence exists in the sample to be detected or not; and secondly, carrying out agarose gel electrophoresis on the amplification product, and judging whether the amplification result is positive or not according to the electrophoresis strip, namely whether the target sequence exists in the sample to be detected or not.
TABLE 4 nucleic acid amplification reaction System of Cronobacter sakazakii (Cronobacters sazakii)
| System of | Volume (μ l) | Final concentration |
| R300 MIX(TaKaRaTaqTM HS Perfect Mix) | 12.5 | / |
| Cro-F/Cro-R(50μM) | 0.25+0.25 | 0.5μM |
| 100％DMSO | 0.5 | 2％ |
| DNA template (10 ng) | 0/1 | / |
| ddH2O | Up to 25 | / |
TABLE 5 List of subjects for nucleic acid amplification reaction-specific detection of Cronobacter sakazakii (Cronobacters sazakii)
| 1. Staphylococcus aureus 21600 | 16. Shigella flexneri 1.1868 |
| 2. Staphylococcus aureus subspecies 1.2465 | 17. Escherichia coli 10738 |
| 3. Staphylococcus epidermidis 1.4260 | 18. Pathogenic escherichia coli 10372 |
| 4. Rhodococcus equi 1.4262 | 19. Diarrhea causing Escherichia coli 10411 |
| 5. Bacillus cereus 1.3760 | 20. Enterotoxigenic Escherichia coli 10415 |
| 6. Bacillus mycoides 21473 | 21. Enterotoxigenic Escherichia coli 10665 |
| 7. Listeria monocytogenes 21635 | 22. Go outEscherichia coli 21530 |
| 8. Listeria lnokhei 10417 | 23. Sakazakii 21560 |
| 9. Listeria monocytogenes 21663 | 24. Yersinia enterocolitica 21669 |
| 10. Salmonella enteritidis subspecies 1.1859 | 25. Yersinia pseudotuberculosis 53504 |
| 11. Salmonella enteritidis 21482 | 26. Vibrio vulnificus 21615 |
| 12. Salmonella typhimurium 10420 | 27. Vibrio parahaemolyticus 1.1997 |
| 13. |
28. Vibrio furiosus 1.1613 |
| 14. Shigella dysenteriae 1.1869 | 29. Vibrio cholerae 1.8676 |
| 15. Shigella boydii 1.10618 | 30. Shigella flexneri |
The experimental results are shown in FIGS. 9, 10 and 11, wherein 1-22 are Staphylococcus aureus, staphylococcus aureus subspecies aureoflavum, staphylococcus epidermidis, rhodococcus equi, bacillus cereus, bacillus mycoides, listeria monocytogenes, listeria inoke, listeria ehelii, salmonella enteritidis, salmonella typhimurium, salmonella paratyphi B, shigella dysenteriae, shigella boydii, shigella flexneri, escherichia coli (containing Clostridium botulinum type A gene), pathogenic Escherichia coli, escherichia coli diarrheal, escherichia enterotoxigenic, escherichia coli enterohemorrhagic, escherichia coli, yersinia enterocolitica, yersinia pseudotuberculosis, vibrio vulnificus, vibrio parahaemolyticus, vibrio franciscensis, vibrio cholerae and Shigella flexneri, respectively, N: negative control, P: positive control (plasmid containing sequence of interest); 23 is cronobacter sakazakii.
FIG. 9 shows the results of the SYBRGreen I dye color development of the present invention for specific detection of nucleic acid amplification reactions against Cronobacter sakazakii (Cronobacters sazakii) at different denaturation temperatures. If the amplification product is bright green, the amplification product is positive; if the amplification product is orange, it is negative. In FIG. 9, the negative control (N) appeared orange when the denaturation temperature was 94 ℃/90 ℃ and was a negative result, as expected; the positive control (P) and cronobacter sakazakii No. 23 exhibited bright green color, which was a positive result, and was as expected. The whole reaction system can work normally. However, the amplified products of the genomic DNA templates of other bacteria showed a great number of positive results, as indicated by tubes 3, 4, 10-18, 20-22, 24, 26, 27, 29 and 30 at a denaturation temperature of 94 ℃ and tubes 3, 7, 10-18, 20-22, 24, 26, 27, 29 and 30 at a denaturation temperature of 90 ℃, which is contrary to the expectation, suggesting that non-specific amplification is likely to occur at a denaturation temperature of 94 ℃/90 ℃ and the results are false positive. When the denaturation temperature is 82 ℃, the negative control (N) is orange, and the negative result is a negative result, which is in line with expectation; the positive control (P) and cronobacter sakazakii No. 23 exhibited bright green color, which was a positive result, and was as expected. The whole reaction system can work normally. Meanwhile, the detection results of the genomic DNA templates of other bacteria are negative, as shown in the No. 1-22 and No. 24-30 tubes. All results were as expected, suggesting that at the denaturation temperature of 82 ℃ no or even a slight amount of non-specific amplification occurred but not enough to affect the dye-based outcome determination.
FIG. 10 shows the results of electrophoresis of amplification products of the nucleic acid amplification reaction of the present invention against Cronobacters sakazakii (Cronobacters sazakii) with specific detection at a denaturation temperature of 94 ℃. If a single band exists at the 291bp position after electrophoresis of the amplification product, the amplification product is positive; if the amplified product has no band after electrophoresis, the amplified product is negative; if one or more bands appear outside 291bp after electrophoresis of the amplification reaction product, the amplification reaction product is false positive caused by non-specific amplification. In FIG. 10, the negative control (N) has no band, as expected; the positive control (P) and the Cronobacter sakazakii No. 23 both have a clear band at 291bp, which is expected. The whole reaction system can work normally. However, the amplified products of the genomic DNA templates of other bacteria showed many bands in the range of 500bp to 2000bp, which is not in line with the expectation, suggesting that the reaction system produced a large amount of non-specific amplification at a denaturation temperature of 94 ℃ and the result is consistent with the dye coloration result in FIG. 9.
FIG. 11 shows the results of electrophoresis of amplification products of the nucleic acid amplification reaction of the present invention against Cronobacters sakazakii (Cronobacters sazakii) which is specifically detected at a denaturation temperature of 82 ℃. If a single band exists at the 291bp position after the electrophoresis of the amplification product, the amplification product is positive; if the amplification reaction product has no band after electrophoresis, the amplification reaction product is negative; if one or more bands appear outside 291bp after electrophoresis of the amplification reaction product, the amplification reaction product is false positive caused by non-specific amplification. In FIG. 11, the negative control (N) had no bands, as expected; the positive control (P) and the Cronobacter sakazakii No. 23 both have a clear band at 291bp, which is expected. The whole reaction system can work normally. Meanwhile, most of the amplification reaction products of the genome DNA templates of other bacteria have no band and show negative results, such as lanes 2-6, 10, 12, 13, 15-22 and 26-30; the products of a few template amplification reactions showed very weak bands after electrophoresis, which were easily distinguished from the positive results, as shown in 1, 7, 8, 9, 11, 14, 24, 25 tubes, indicating that no or very little non-specific amplification was produced at the denaturation temperature of 82 ℃ which was easily distinguished in the electrophoresis results.
As can be seen from fig. 9-11 and table 5, the nucleic acid amplification method of the present invention has good strain specificity for the application of cronobacter sakazakii, i.e., only cronobacter sakazakii exhibits a positive result, but not cronobacter sakazakii exhibits a negative result, under the reaction system and reaction conditions proposed in the present invention. It is to be noted that, when the reaction is carried out at the denaturation temperature (90 ℃ or 94 ℃) of the conventional PCR method, although the electrophoresis result (FIG. 10) shows that the target detection object (Cronobacter sakazakii, lane 23) has a single clear band, the non-target detection object generally undergoes a large amount of non-specific amplification, such as Staphylococcus epidermidis in lane 3; when the detection result was judged by the dye-color development method, as shown in FIG. 9, a large number of false positive results were obtained under the denaturing conditions of 90 ℃ and 94 ℃, and thus the detection object and the non-detection object could not be distinguished from each other. In the same reaction system, when the reaction is carried out at the denaturation temperature of 82 ℃ calculated by the method, the electrophoresis result (figure 11) shows that a target detection object (cronobacter sakazakii, lane 23) has a single clear band, while non-target detection objects have less non-specific amplification, and even if the non-target detection objects have the single clear band, the non-specific amplification band is weak and is easily distinguished from a positive result; when the detection result was judged by the dye-color method, as shown in fig. 9, only the target detection target (cronobacter sakazakii, tube No. 23) gave a positive result and all the non-target detection targets gave a negative result under the denaturation condition at 82 ℃.
Example 3 Simultaneous detection of Salmonella, staphylococcus aureus, yersinia enterocolitica and Cronobacter sakazakii
The genome targeted by the primer design in the invention is changed into the genomes of salmonella, staphylococcus aureus, yersinia enterocolitica and cronobacter sakazakii, AT-rich sequences are screened, specific primers are designed, primer combinations are screened according to the calculated primer Tm and the target sequence Tm, reaction conditions are set for nucleic acid amplification, whether the target sequence exists in a sample to be detected is determined by judging whether the reaction result is positive, and further whether one or more of salmonella, staphylococcus aureus, yersinia enterocolitica and cronobacter sakazakii exist in the sample to be detected is determined. The method comprises the following specific steps:
(1) Screening of target sequences rich in AT bases:
2896 total genome sequences were used for the bacteria, archaea and virus data with complete genome sequences downloaded from FTP at NCBI, 8/5 in 2019. Set a, which contains all salmonella genomic sequences; set B was set, which contained all non-salmonella genomic sequences. The salmonella genome sequence is used as a reference genome, a window with the width of 1000bp slides from the first base of the genome, and the step length is 50bp; calculating the base content of the sequence AT once before each sliding, and reserving a region with the base content of the sequence AT more than 60% as a candidate target sequence. The above process is implemented using perl scripts.
(2) Design of specific primers:
according to the characteristics of the PCR primer and the requirements of the invention, the characteristic parameters of the primer, such as the AT base content of 55-75%, the 3 'end stability delta G <4, the 5' end stability delta G <3, the primer sequence length (20-36 bp) and the like, are set, meanwhile, the conditions that a single primer cannot generate a hairpin structure and cannot interact with the single primer are set, and the single primer meeting the set conditions is calculated by taking the candidate target sequence in the step (1) as a candidate sequence for designing the single primer. The Tm value of a single primer is calculated by the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length), and the position of each primer on the target sequence, the positive and negative strand information (i.e. whether it is from the positive strand or the negative strand), and the length of the primer are recorded.
And (3) carrying out primer pairing according to the position information of the single primer, and reserving a primer pair which simultaneously meets the conditions that the Tm difference of the primer pair is less than 3 ℃ and the amplification area of the primer pair is between 200 and 600bp as a candidate primer pair.
And (3) using alignment software Bowtie to perform sequence alignment on each primer in the candidate primer pair designed in the last step with the target genome sequence in the set A and the non-target genome sequence in the set B respectively. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in the set A, the parameter setting of '-a-n 0' is used, namely the single primer is required to be completely matched with the target sequence; to ensure primer specificity, a parameter setting of "-a-n 3" was used when the single primer was aligned to the non-target sequences in pool B, i.e., the single primer was allowed no more than 3 mismatches to the non-target sequences. The system outputs primer pairs satisfying the condition, in which the number of primer pairs can be set in advance, and in this embodiment, the number of primer pairs is set in advance to 100. The high-throughput and automatic primer design process is realized by adopting C and Perl scripts. After the program is operated, 100 salmonella amplification primer pairs are designed in the AT enrichment region.
100 amplification primer pairs of staphylococcus aureus, yersinia enterocolitica and cronobacter sakazakii are respectively designed by the same method. Calculating Tm of each primer using the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length) and calculating the average Tm value of the primer pair as the reaction annealing temperature; the denaturation temperature of the amplified region of each primer pair was calculated using the formula 0.357X (percent GC). Times.100 +70.582- (990/amplification product sequence length).
Screening the primer combination by taking the conditions that the difference of the amplification sequence lengths of different bacteria is at least 50bp, the reaction denaturation temperature is at most 2 ℃ and the reaction annealing temperature is at most 2 ℃. The system outputs primer combinations satisfying the condition, and the number thereof may be set in advance, and in this embodiment, the number of primer combinations is set in advance to 10. And randomly selecting a primer group for validity verification. The sequences of the primer combination are as follows:
primer pair A:
Sal-F1：5’-TCAGACATCCGTTCAGAAAAT-3’(SEQ ID NO.1)
Sal-R1：5’-GTTCAACTGTCGACAAGATTAA-3’(SEQ ID NO.2)
and (3) primer pair B:
Sta-F：5’-GTAGGTATGGTAAATAGTTACAC-3’(SEQ ID NO.5)
Sta-R:5’-CACTAATGCCAAATTTACTTAAAATCG-3’(SEQ ID NO.6)
and (3) primer pair C:
Cro-F：5’-CGCCATAACTGCATAATCAT-3’(SEQ ID NO.3)
Cro-R:5’-ATAACGAGTTACCGTGCAGA-3’(SEQ ID NO.4)
and (3) primer pair D:
Yer-F：5’-TGTGCGGTGGATGTAAATAATTC-3’(SEQ ID NO.7)
Yer-R:5’-GCTTTGAAACTCAAGGACTG-3’(SEQ ID NO.8)
the AT base content of the primer pair A is 60 percent and 64 percent respectively, and the theoretical average annealing temperature is 62 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 355bp, the percentage content of AT is 71%, and the theoretical denaturation temperature is 78.14 ℃.
The AT base content of the primer pair B is 65.22 percent and 70.37 percent respectively, and the theoretical average annealing temperature is 62.5 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 465bp, the percentage content of AT is 73.76%, and the theoretical denaturation temperature is 77.82 ℃.
The AT base content of the primer pair C is 60 percent and 55 percent respectively, and the theoretical average annealing temperature is 63 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 291bp, the percentage content of AT is 68.73%, and the theoretical denaturation temperature is 78.34 ℃.
The AT base content of the primer pair D is 60.87 percent and 55 percent respectively, and the theoretical average annealing temperature is 64 ℃; the length of the theoretical amplification product sequence fragment of the primer pair is 235bp, the percentage content of AT is 68.09%, and the theoretical denaturation temperature is 77.76 ℃.
(4) Nucleic acid amplification reaction and result detection:
the nucleic acid amplification reaction system configuration is shown in Table 6 below. According to the calculation results, the above process is recommended to be repeated 35 times by using denaturation at 81 ℃ for 5 seconds, annealing at 62 ℃ for 5 seconds and elongation at 72 ℃ for 20 seconds. The list of test subjects is shown in Table 7.
After the reaction is finished by using a common gradient PCR instrument, the amplification result can be judged by two modes, firstly, SYBRGreen I dye with the final concentration of 25x is added, and whether the amplification result is positive or not is judged by color, namely whether a target sequence exists in a sample to be detected or not is judged; and secondly, carrying out agarose gel electrophoresis on the amplification product, and judging whether the amplification result is positive or not according to the electrophoresis strip, namely whether the target sequence exists in the sample to be detected or not.
TABLE 6 four bacterial nucleic acid amplification reaction systems
TABLE 7 list of four bacteria nucleic acid amplification reaction-specific detection targets
| 1. Staphylococcus aureus 21600 | 16. Shigella flexneri 1.1868 |
| 2. Staphylococcus aureus subspecies 1.2465 | 17. Escherichia coli 10738 |
| 3. Staphylococcus epidermidis 1.4260 | 18. Pathogenic escherichia coli 10372 |
| 4. Rhodococcus equi 1.4262 | 19. Diarrhea causing Escherichia coli 10411 |
| 5. Bacillus cereus 1.3760 | 20. Enterotoxigenic Escherichia coli 10415 |
| 6. Bacillus mycoides 21473 | 21. Enterotoxigenic Escherichia coli 10665 |
| 7. Listeria monocytogenes 21635 | 22. Hemorrhagic Escherichia coli 21530 |
| 8. Listeria lnokhei 10417 | 23. Cronobacter sakazakii 21560 |
| 9. Listeria monocytogenes 21663 | 24. Yersinia enterocolitica 21669 |
| 10. Salmonella enteritidis subspecies 1.1859 | 25. Yersinia pseudotuberculosis 53504 |
| 11. Salmonella enteritidis 21482 | 26. Vibrio vulnificus 21615 |
| 12. Salmonella typhimurium 10420 | 27. Vibrio parahaemolyticus 1.1997 |
| 13. Paratyphoid B salmonella 10437 | 28. Vibrio frenuli 1.1613 |
| 14. Shigella dysenteriae 1.1869 | 29. Vibrio cholerae 1.8676 |
| 15. Shigella bodyii 1.10618 | 30. Shigella sonnei |
FIGS. 12 and 13 show the results of the fluorescent dye staining and the agarose gel electrophoresis of the nucleic acid amplification reaction of the present invention against four bacteria, salmonella enteritidis subspecies, staphylococcus aureus subspecies, yersinia enterocolitica, cronobacter sakazakii. Wherein, N is the corresponding negative template amplification result, M1 refers to Marker DL2000, M2 refers to Marker B (100-600 bp), and mixed refers to the mixed template of four bacterial genome DNAs. FIG. 12 shows that the results of the single-strain, mixed-template and negative-template amplification and color development are expected. FIG. 13 shows that each single amplified fragment of the bacteria meets the theoretical calculation and that each amplified fragment of the bacteria of the mixed template species can be separated from each other.
FIG. 14 shows the SYBRGreen I dye color development results of the specific detection of the nucleic acid amplification reaction of the present invention against four bacteria, salmonella, staphylococcus aureus, yersinia enterocolitica, and Cronobacter sakazakii, at a denaturation temperature of 81 ℃. If the amplification product is bright green, the amplification product is positive; if the amplification product is orange, it is negative. In fig. 14, staphylococcus aureus No. 1-2, staphylococcus aureus subspecies, salmonella enteritidis subspecies No. 10-13, salmonella enteritidis, salmonella typhimurium and salmonella paratyphi b, salmonella sakazakii No. 23, yersinia enterocolitica No. 24, appeared bright green, and were positive results, which were expected; meanwhile, the detection results of the genomic DNA templates of other bacteria are negative, as shown in the tubes 3-9, 14-22 and 25-30. All results were as expected, suggesting that no or even a slight amount of non-specific amplification occurred at the denaturation temperature of 81 ℃ but not enough to affect the dye-based outcome determination.
Example 4 Simultaneous detection of Salmonella and Staphylococcus aureus
The method comprises the steps of screening sequences rich in AT and designing specific primers aiming AT salmonella and staphylococcus aureus genomes, screening primer combinations according to the calculated primer Tm and the target sequence Tm, setting reaction conditions for nucleic acid amplification, determining whether the target sequence exists in a sample to be detected or not by judging whether a reaction result is positive or not, and further determining whether one or more of salmonella and staphylococcus aureus exists in the sample to be detected or not. The method comprises the following specific steps:
(1) Screening of target sequences rich in AT bases:
2896 whole genome sequences were used for bacteria, archaea and virus data with complete genome sequences downloaded from the FTP at NCBI at 8/5 in 2019. Set a, which contains all salmonella genomic sequences; set B was set, which contained all non-salmonella genomic sequences. The salmonella genome sequence is used as a reference genome, a window with the width of 1000bp slides from the first base of the genome, and the step length is 50bp; calculating the base content of the sequence AT once before each sliding, and reserving a region with the base content of the sequence AT more than 60% as a candidate target sequence. The above process is implemented using perl scripts.
(2) Design of specific primers:
according to the characteristics of the PCR primer and the requirements of the invention, the characteristic parameters of the primer, such as the AT base content of 55-75%, the stability delta G AT the 3 'end of less than 4, the stability delta G AT the 5' end of less than 3, the length of the primer sequence (20-36 bp) and the like, are set, meanwhile, the conditions that a single primer cannot generate a hairpin structure, cannot generate interaction per se and the like are set, the candidate target sequence in the step (1) is taken as the candidate sequence for designing the single primer, and the single primer meeting the set conditions is calculated. The Tm value of a single primer is calculated by the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length), and the position of each primer on the target sequence, the positive and negative strand information (i.e. whether it is from the positive strand or the negative strand), and the length of the primer are recorded.
And (3) carrying out primer pairing according to the position information of the single primer, and reserving a primer pair which simultaneously meets the two conditions that the Tm difference of the primer pair is less than 3 ℃ and the amplification area of the primer pair is 200-600 bp as a candidate primer pair.
And (3) using alignment software Bowtie to perform sequence alignment on each primer in the candidate primer pair designed in the last step with the target genome sequence in the set A and the non-target genome sequence in the set B respectively. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in the set A, the parameter setting of '-a-n 0' is used, namely the single primer is required to be completely matched with the target sequence; to ensure primer specificity, a parameter setting of "-a-n 3" was used when the single primer was aligned to the non-target sequences in pool B, i.e., the single primer was allowed no more than 3 mismatches to the non-target sequences. The system outputs primer pairs satisfying the condition, in which the number of primer pairs can be set in advance, and in this embodiment, the number of primer pairs is set in advance to 100. The high-throughput and automatic primer design process is realized by adopting C and Perl scripts. After the program is operated, 100 salmonella amplification primer pairs are designed in the AT enrichment region.
100 staphylococcus aureus amplification primer pairs are respectively designed by the same method. Calculating Tm of each primer using the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length) and calculating the average Tm value of the primer pair as the reaction annealing temperature; the denaturation temperature of the amplified region of each primer pair was calculated using the formula 0.357X (percent GC). Times.100 +70.582- (990/amplification product sequence length).
Screening the primer combination by taking the conditions that the difference of the amplification sequence lengths of different bacteria is at least 50bp, the reaction denaturation temperature is at most 2 ℃ and the reaction annealing temperature is at most 2 ℃. The system outputs primer combinations satisfying the condition, and the number thereof may be set in advance, and in this embodiment, the number of primer combinations is set in advance to 10. One primer set was randomly selected for validation. The sequences of the primer combination are as follows:
and (3) primer pair A:
Sal-F1：5’-TCAGACATCCGTTCAGAAAAT-3’(SEQ ID NO.1)
Sal-R1：5’-GTTCAACTGTCGACAAGATTAA-3’(SEQ ID NO.2)
and a primer pair E:
Sta-F2：5’-CCTTTCATCTAAAAACCTCCA-3’(SEQ ID NO.9)
Sta-R2:5’-GAAATGGATGTTTTAAAAGAAGG-3’(SEQ ID NO.10)
the AT base content of the primer pair A is 60 percent and 64 percent respectively, and the theoretical average annealing temperature is 62 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 355bp, the percentage content of AT is 71%, and the theoretical denaturation temperature is 78.14 ℃.
The AT base content of the primer pair E is 61.90 percent and 69.57 percent respectively, and the theoretical average annealing temperature is 61 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 595bp, the percentage content of AT is 76.30%, and the theoretical denaturation temperature is 77.38 ℃.
(4) Nucleic acid amplification reaction and result detection:
the nucleic acid amplification reaction system configuration is shown in Table 8 below. According to the calculation results, the above process is recommended to be repeated 35 times by using denaturation at 81 ℃ for 5 seconds, annealing at 62 ℃ for 5 seconds and elongation at 72 ℃ for 20 seconds. The list of test subjects is shown in Table 7.
After the reaction is finished by using a common gradient PCR instrument, the amplification product is subjected to agarose gel electrophoresis, and whether the amplification result is positive or not is judged according to an electrophoresis strip, namely whether a target sequence exists in a sample to be detected or not is judged.
TABLE 8 two bacterial nucleic acid amplification reaction systems
FIG. 15 shows the result of agarose gel electrophoresis of the nucleic acid amplification reaction of the present invention against two bacteria, salmonella enteritidis subspecies and Staphylococcus aureus subspecies. Wherein, N is the amplification result of the corresponding negative template, M is Marker DL2000, salmonella is Salmonella enteritidis subspecies, staphylococcus aureus subspecies, and mixed template of the two bacterial genome DNAs. FIG. 15 shows that each single-strain amplified fragment is in accordance with the theoretical calculation value and each strain amplified fragment of the mixed template can be separated from each other.
Example 5 Simultaneous detection of Salmonella and Yersinia enterocolitica
The method comprises the steps of screening AT-rich sequences and designing specific primers aiming AT salmonella and yersinia enterocolitica genomes, screening primer combinations according to calculated primer Tm and target sequence Tm, setting reaction conditions for nucleic acid amplification, determining whether a target sequence exists in a sample to be detected by judging whether a reaction result is positive, and further determining whether one or more of salmonella and yersinia enterocolitica exists in the sample to be detected. The method comprises the following specific steps:
(1) Screening of target sequences rich in AT bases:
2896 whole genome sequences were used for bacteria, archaea and virus data with complete genome sequences downloaded from the FTP at NCBI at 8/5 in 2019. Set a, which contains all salmonella genomic sequences; set B was set, which contained all non-salmonella genomic sequences. The salmonella genome sequence is used as a reference genome, a window with the width of 1000bp slides from the first base of the genome, and the step length is 50bp; calculating the base content of the sequence AT once before each sliding, and reserving a region with the base content of the sequence AT more than 60% as a candidate target sequence. The above process is implemented by using perl script.
(2) Design of specific primers:
according to the characteristics of the PCR primer and the requirements of the invention, the characteristic parameters of the primer, such as the AT base content of 55-75%, the 3 'end stability delta G <4, the 5' end stability delta G <3, the primer sequence length (20-36 bp) and the like, are set, meanwhile, the conditions that a single primer cannot generate a hairpin structure and cannot interact with the single primer are set, and the single primer meeting the set conditions is calculated by taking the candidate target sequence in the step (1) as a candidate sequence for designing the single primer. The Tm value of a single primer is calculated by the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length), and the position of each primer on the target sequence, the plus and minus chain information (i.e., whether derived from the plus chain or the minus chain), and the length of the primer are recorded.
And (3) carrying out primer pairing according to the position information of the single primer, and reserving a primer pair which simultaneously meets the conditions that the Tm difference of the primer pair is less than 3 ℃ and the amplification area of the primer pair is between 200 and 600bp as a candidate primer pair.
And (3) using alignment software Bowtie to perform sequence alignment on each primer in the candidate primer pair designed in the last step with the target genome sequence in the set A and the non-target genome sequence in the set B respectively. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in the set A, the parameter setting of '-a-n 0' is used, namely the single primer is required to be completely matched with the target sequence; to ensure primer specificity, when the single primer is aligned to the non-target sequences in pool B, a parameter setting of "-a-n 3" is used, i.e., the single primer is allowed to have no more than 3 mismatches to the non-target sequences. The system outputs primer pairs satisfying the condition, in which the number of primer pairs can be set in advance, and in this embodiment, the number of primer pairs is set in advance to 100. The high-throughput and automatic primer design process is realized by adopting C and Perl scripts. After the program is operated, 100 salmonella amplification primer pairs are designed in the AT enrichment region.
100 Yersinia enterocolitica amplification primer pairs are designed by the same method. Calculating Tm of each primer using the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length) and calculating the average Tm value of the primer pair as the reaction annealing temperature; the denaturation temperature of the amplified region of each primer pair was calculated using the formula 0.357X (percent GC content). Times.100 +70.582- (990/amplification product sequence length).
Screening the primer combination by taking the conditions that the difference of the amplification sequence lengths of different bacteria is at least 50bp, the reaction denaturation temperature is at most 2 ℃ and the reaction annealing temperature is at most 2 ℃. The system outputs primer combinations satisfying the condition, and the number thereof may be set in advance, and in this embodiment, the number of primer combinations is set in advance to 10. One primer set was randomly selected for validation. The sequences of the primer combination are as follows:
and (3) primer pair F:
Sal-F2：5’-TGGGTTGAAATAGCCCATTA-3’(SEQ ID NO.11)
Sal-R2：5’-GACGTGACACACTTCGTTTT-3’(SEQ ID NO.12)
and (3) primer pair G:
Yer-F2：5’-ATGGAAAATAACATAATTTCTATTACCGG-3’(SEQ ID NO.13)
Yer-R2:5’-TCTCTGCGAATAACCTTGTG-3’(SEQ ID NO.14)
the AT base content of the primer pair F is 60 percent and 55 percent respectively, and the theoretical average annealing temperature is 63 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 352bp, the percentage content of AT is 74.43%, and the theoretical denaturation temperature is 76.90 ℃.
The AT base content of the primer pair G is 72.41 percent and 55 percent respectively, and the theoretical average annealing temperature is 63.5 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 242bp, the percentage content of AT is 72.02%, and the theoretical denaturation temperature is 76.48 ℃.
(4) Nucleic acid amplification reaction and result detection:
the nucleic acid amplification reaction system configuration is shown in Table 9 below. According to the calculation results, the above process is recommended to be repeated 35 times by using denaturation at 81 ℃ for 5 seconds, annealing at 62 ℃ for 5 seconds and elongation at 72 ℃ for 20 seconds. The list of test subjects is shown in Table 7.
After the reaction is finished by using a common gradient PCR instrument, the amplification product is subjected to agarose gel electrophoresis, and whether the amplification result is positive or not is judged according to an electrophoresis strip, namely whether a target sequence exists in a sample to be detected or not is judged.
TABLE 9 two bacterial nucleic acid amplification reaction systems
| System of | Volume (μ l) | Final |
| Taq R300mix | ||
| 15 | 1x | |
| SAL-F2/R2(50μM) | 0.25+0.25 | 0.5μM |
| YER-F2/R2(50μM) | 0.25+0.25 | 0.5μM |
| 100％DMSO | 0.5 | 2％ |
| Stencil (10 ng) | 0/1 | / |
| ddH2O | Upto25 | / |
FIG. 16 shows the result of agarose gel electrophoresis of the nucleic acid amplification reaction of the present invention against two bacteria, salmonella enteritidis subspecies and Yersinia enterocolitica. Wherein "N" is the amplification result of the corresponding negative template, "M" refers to Marker DL2000, "Salmonella refers to Salmonella enteritidis subspecies," Yersi "refers to Yersinia enteronitis subspecies, and" mixed "refers to the mixed template of the genomic DNA of the two species of bacteria. FIG. 16 shows that each single-strain amplified fragment is in accordance with the theoretical calculation value and each strain amplified fragment of the mixed template can be separated from each other.
Example 6 Simultaneous detection of Salmonella, staphylococcus aureus, and Yersinia enterocolitica
The method comprises the steps of screening sequences rich in AT aiming AT genomes of salmonella, staphylococcus aureus and yersinia enterocolitica, designing specific primers, screening primer combinations according to calculated primer Tm and target sequence Tm, setting reaction conditions for nucleic acid amplification, determining whether a target sequence exists in a sample to be detected by judging whether a reaction result is positive, and further determining whether one or more of salmonella, staphylococcus aureus and yersinia enterocolitica exist in the sample to be detected. The method comprises the following specific steps:
(1) Screening of target sequences rich in AT bases:
2896 total genome sequences were used for the bacteria, archaea and virus data with complete genome sequences downloaded from FTP at NCBI, 8/5 in 2019. Set a, which contains all salmonella genomic sequences; set B was set, which contained all non-salmonella genomic sequences. The salmonella genome sequence is used as a reference genome, a window with the width of 1000bp slides from the first base of the genome, and the step length is 50bp; calculating the base content of the sequence AT once before each sliding, and reserving a region with the base content of the sequence AT more than 60% as a candidate target sequence. The above process is implemented using perl scripts.
(2) Design of specific primers:
according to the characteristics of the PCR primer and the requirements of the invention, the characteristic parameters of the primer, such as the AT base content of 55-75%, the stability delta G AT the 3 'end of less than 4, the stability delta G AT the 5' end of less than 3, the length of the primer sequence (20-36 bp) and the like, are set, meanwhile, the conditions that a single primer cannot generate a hairpin structure, cannot generate interaction per se and the like are set, the candidate target sequence in the step (1) is taken as the candidate sequence for designing the single primer, and the single primer meeting the set conditions is calculated. The Tm value of a single primer is calculated by the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length), and the position of each primer on the target sequence, the plus and minus chain information (i.e., whether derived from the plus chain or the minus chain), and the length of the primer are recorded.
And (3) carrying out primer pairing according to the position information of the single primer, and reserving a primer pair which simultaneously meets the conditions that the Tm difference of the primer pair is less than 3 ℃ and the amplification area of the primer pair is between 200 and 600bp as a candidate primer pair.
And (3) using alignment software Bowtie to perform sequence alignment on each primer in the candidate primer pair designed in the last step with the target genome sequence in the set A and the non-target genome sequence in the set B respectively. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in the set A, the parameter setting of '-a-n 0' is used, namely the single primer is required to be completely matched with the target sequence; to ensure primer specificity, a parameter setting of "-a-n 3" was used when the single primer was aligned to the non-target sequences in pool B, i.e., the single primer was allowed no more than 3 mismatches to the non-target sequences. The system outputs primer pairs satisfying the condition, in which the number of primer pairs can be set in advance, and in this embodiment, the number of primer pairs is set in advance to 100. The high-throughput and automatic primer design process is realized by adopting C and Perl scripts. After the program is operated, 100 salmonella amplification primer pairs are designed in the AT enrichment region.
100 Yersinia enterocolitica amplification primer pairs are respectively designed by the same method. Calculating Tm of each primer using the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length) and calculating the average Tm value of the primer pair as the reaction annealing temperature; the denaturation temperature of the amplified region of each primer pair was calculated using the formula 0.357X (percent GC). Times.100 +70.582- (990/amplification product sequence length).
Screening the primer combination by taking the conditions that the difference of the amplification sequence lengths of different bacteria is at least 50bp, the reaction denaturation temperature is at most 2 ℃ and the reaction annealing temperature is at most 2 ℃. The system outputs primer combinations satisfying the condition, and the number may be set in advance, and in this embodiment, the number of primer combinations is set in advance to 10. And randomly selecting a primer group for validity verification. The sequences of the primer combination are as follows:
primer pair A:
Sal-F1：5’-TGGGTTGAAATAGCCCATTA-3’(SEQ ID NO.1)
Sal-R1：5’-GACGTGACACACTTCGTTTT-3’(SEQ ID NO.2)
and (3) primer pair D:
Yer-F：5’-ATGGAAAATAACATAATTTCTATTACCGG-3’(SEQ ID NO.7)
Yer-R:5’-TCTCTGCGAATAACCTTGTG-3’(SEQ ID NO.8)
and (3) primer pair E:
Sta-F2：5’-CCTTTCATCTAAAAACCTCCA-3’(SEQ ID NO.9)
Sta-R2:5’-GAAATGGATGTTTTAAAAGAAGG-3’(SEQ ID NO.10)
the AT base content of the primer pair A is 60 percent and 55 percent respectively, and the theoretical average annealing temperature is 63 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 352bp, the percentage content of AT is 74.43 percent, and the theoretical denaturation temperature is 76.90 ℃.
The AT base content of the primer pair D is 72.41 percent and 55 percent respectively, and the theoretical average annealing temperature is 63.5 ℃; the theoretical amplification product sequence fragment length of the primer pair is 242bp, the percentage content of AT is 72.02%, and the theoretical denaturation temperature is 76.48 ℃.
The AT base content of the primer pair E is 61.90 percent and 69.57 percent respectively, and the theoretical average annealing temperature is 61 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 595bp, the percentage content of AT is 76.30%, and the theoretical denaturation temperature is 77.38 ℃.
(4) Nucleic acid amplification reaction and result detection:
the nucleic acid amplification reaction system configuration is shown in Table 10 below. According to the calculation results, the above process is recommended to be repeated 35 times by using denaturation at 81 ℃ for 5 seconds, annealing at 62 ℃ for 5 seconds and elongation at 72 ℃ for 20 seconds. The list of test subjects is shown in Table 7.
After the reaction is finished by using a common gradient PCR instrument, the amplification product is subjected to agarose gel electrophoresis, and whether the amplification result is positive or not is judged according to the electrophoresis band, namely whether the target sequence exists in the sample to be detected or not is judged.
TABLE 10 nucleic acid amplification reaction System for three bacteria
| System of | Volume (μ l) | Final |
| Taq R300mix | ||
| 15 | 1x | |
| SAL-F/R(50μM) | 0.25+0.25 | 0.5μM |
| YER-F/R(50μM) | 0.25+0.25 | 0.5μM |
| STA-F2/R2(50μM) | 0.25+0.25 | 0.5μM |
| 100％DMSO | 0.5 | 2％ |
| Stencil (10 ng) | 0/1 | / |
| ddH2O | Upto25 | / |
FIG. 17 shows the results of agarose gel electrophoresis of nucleic acid amplification reactions of three bacteria of Salmonella enteritidis subspecies, staphylococcus aureus subspecies, and Yersinia enterocolitica according to the present invention. Wherein "N" is the amplification result of the corresponding negative template, "M" refers to Marker DL2000, "Enteria" refers to Salmonella enteritidis subspecies, "Small Ye" refers to Yersinia enteronitis subspecies, "Kinia" refers to Staphylococcus aureus subspecies, and "Mixed" refers to the mixed template of the genomic DNAs of the three bacteria. FIG. 17 shows that each single amplified fragment of the bacteria meets the theoretical calculation and that each amplified fragment of the bacteria of the mixed template species can be separated from each other.
Reference to the literature
Zhou Hui Qi (2014) relationship between GC content of genome and usage preference of base, codon and amino acid (master master), university of electronic technology (67)
The protection content of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.
SEQUENCE LISTING
<110> Shanghai bioinformatics technology research center, wangwang food group Co., ltd
<120> method for detecting salmonella, application and primer thereof
<160> 15
<170> PatentIn version 3.3
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tgtgcggtgg atgtaaataa ttc 23
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence
<400> 8
<210> 9
<211> 21
<212> DNA
<213> Artificial sequence
<400> 9
cctttcatct aaaaacctcc a 21
<210> 10
<211> 23
<212> DNA
<213> Artificial sequence
<400> 10
gaaatggatg ttttaaaaga agg 23
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence
<400> 11
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence
<400> 12
<210> 13
<211> 29
<212> DNA
<213> Artificial sequence
<400> 13
atggaaaata acataatttc tattaccgg 29
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence
<400> 14
<210> 15
<211> 19
<212> DNA
<213> Artificial sequence
<400> 15
aagggggttc caggcatta 19
Claims (13)
1. A method for detecting salmonella comprising the steps of:
(1) Screening a target sequence rich in AT base in a salmonella genome sequence;
(2) Designing a primer with both universality and specificity aiming at the target sequence;
(3) Calculating the inverse strain temperature and the annealing temperature based on the percentage content of GC, the length of the primer sequence and the length of the theoretical amplification product sequence of the primer pair, and setting the reaction conditions of nucleic acid amplification;
(4) And carrying out nucleic acid amplification reaction under the condition of local melting of the template to obtain an amplification product.
2. The method of claim 1, wherein the primer pair is:
and (3) primer pair A:
Sal-F1：5’-TGGGTTGAAATAGCCCATTA-3’(SEQ ID NO.1)
Sal-R1：5’-GACGTGACACACTTCGTTTT-3’(SEQ ID NO.2)。
3. the method of claim 1, wherein in step (1), the salmonella genomic sequence is slid over a window of 1000bp in width starting from the first base by 5-100 bp; calculating the AT base content of the sequence contained in the position of each window, and reserving the region with the AT base content of the sequence more than 60 percent as the target sequence.
4. The method of claim 1, wherein in step (2), the primer is designed by a method comprising: (2.1) designing a single primer aiming at a target sequence to obtain a candidate primer; (2.2) judging the physicochemical property of the candidate primer, and screening a single primer meeting the requirement; (2.3) combining the single primers obtained by screening in the step (2.2) into a primer pair; (2.4) judging the universality and the specificity of the primer pair; and (2.5) outputting a primer pair meeting the conditions to obtain the specific primer.
5. The method of claim 4, wherein in step (2.1), the candidate primers satisfy the following condition: a) The length of the primer sequence is between 20bp and 36 bp; b) The AT base content is 55-80%; c) The continuous GC number is less than or equal to 5; simultaneously recording position information and positive and negative chain information of the candidate primers matched on the target sequence; and/or, in the step (2.2), judging the physicochemical property of the single primer which meets the condition (2.1), wherein the physicochemical property comprises 3 'end stability, 5' end stability and/or secondary structure stability.
6. The method according to claim 4, wherein in the step (2.3), the single primers obtained by screening in the step (2.2) are combined into a primer pair according to the position information and the positive and negative chain information of the single primers matched with the target sequence; the length of the theoretical amplification product sequence of the primer pair is between 200bp and 600 bp; calculating Tm value of single primer according to formula 0.466X (GC percentage content) 100+66.04- (450/primer sequence length); screening the primer pairs under the conditions that the Tm difference value of the primer pairs is less than or equal to 3 ℃ and the primers cannot interact with each other to obtain candidate primer pairs;
wherein, the percentage content of GC refers to the percentage of the number of bases G and C in the primer to the total number of bases of the primer;
the length of the primer sequence refers to the number of bases of the primer.
7. The method of claim 4, wherein in step (2.4), the commonality determination is a check of whether the primer pairs exactly match all target sequences; the specificity determination means to examine whether or not a single primer cannot be specifically matched with a non-target sequence other than all target sequences, i.e., nucleic acid sequences other than the nucleic acid sequence to be amplified, and the specific match means a match of not more than 2 mismatches.
8. The method as claimed in claim 1, wherein in step (3), the reaction denaturation temperature is calculated as 0.357 x (GC percentage) x 100+70.582- (990/primer pair theoretical amplification product sequence length) and is recorded as Tma; wherein, the percentage content of GC is the percentage of the number of bases G and C in the theoretical amplification product sequence of the primer pair to the total number of bases of the theoretical amplification product sequence of the primer pair; and/or the presence of a gas in the atmosphere,
calculating the Tm value of a single primer according to a formula of 0.466 multiplied by 100+66.04- (450/primer sequence length of GC percentage content), and taking the average value of the Tm value to obtain the reaction annealing temperature which is marked as Tmb; wherein, the percentage content of GC refers to the percentage of the number of bases G and C in the primer to the total number of bases of the primer.
9. The method of claim 8, wherein in step (3), the nucleic acid amplification reaction conditions comprise: reacting at the denaturation temperature for 5 seconds, at the annealing temperature for 5 seconds, and at the extension temperature for 20 seconds, wherein the above processes are repeated for 30-40 times; wherein the denaturation temperature can be adjusted between Tma + -5 ℃ as required; the annealing temperature can be adjusted between Tmb +/-2 ℃ according to requirements; the extension temperature was 72 ℃.
10. Primer pairs obtainable by the method according to any one of claims 1 to 9.
11. A diagnostic reagent comprising the primer pair of claim 10.
12. Use of the method of any one of claims 1 to 9 for nucleic acid amplification of a target nucleic acid sequence enriched in AT regions, use of the method of any one of claims 1 to 9 or of the primer pair of claim 10 for amplification and/or detection of a salmonella-related gene or region, use of the method of any one of claims 1 to 9 or of the primer pair of claim 10 for the preparation of a product for amplification and/or detection of a salmonella-genome-related gene or region, use of the diagnostic reagent of claim 11 for detection of salmonella.
13. Use of the method of any one of claims 1 to 9 for the simultaneous detection of any two or more of salmonella, staphylococcus aureus, cronobacter sakazakii, yersinia enterocolitica.
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