CN110734988B - A method for isothermal amplification of methicillin-resistant Staphylococcus aureus (MRSA) nucleic acid - Google Patents

Abstract

The invention discloses a method for isothermal amplification of methicillin-resistant Staphylococcus aureus (MRSA) nucleic acid, specifically, the method of the invention comprises the steps of: amplification in a reaction system, wherein the reaction system contains two specific primer pairs for amplifying methicillin-resistant Staphylococcus aureus (MRSA), and the primers can amplify the amplification products of two characteristic sequences (SA 23s rRNA and mecA RNA) corresponding to methicillin-resistant Staphylococcus aureus (MRSA) from a test sample with an extremely low MRSA copy number. The method of the invention can amplify the sample to be tested containing MRSA (SA 23s rRNA and mecA RNA) with high specificity, high sensitivity, low pollution, and rapidity, and has the characteristics of high detection efficiency and high accuracy, and has broad application prospects.

Description

A method for isothermal amplification of methicillin-resistant Staphylococcus aureus (MRSA) nucleic acid

Technical Field

The present invention relates to the technical field of bacterial biological detection, and in particular to primers, probes and related kits used in real-time fluorescent nucleic acid isothermal amplification detection of methicillin-resistant Staphylococcus aureus (MRSA) by combining specific target capture technology with real-time fluorescent nucleic acid isothermal amplification detection technology.

Background technique

Staphylococcus aureus (SA) is an important pathogen in medicine. It is widely distributed, has various infection types, and has a high drug resistance rate. In particular, methicillin-resistant Staphylococcus aureus (MRSA) infection is multi-drug resistant and has a high mortality rate, which has become a difficulty in clinical anti-infection treatment. Staphylococcus aureus is the most common pathogen in human suppurative infections. It can cause local suppurative infections, pneumonia, pseudomembranous colitis, pericarditis, etc., and even serious systemic infections such as sepsis and septicemia.

Jevons first discovered MRSA in the UK in 1961. In the mid-1960s, it spread to many European countries and Canada. In the late 1970s, it increased rapidly throughout the world and caused an epidemic, leading to an increase in mortality and medical expenses, becoming a global problem. In the 1990s, research reports on the prevalence of MRSA increased significantly around the world. In some large teaching hospitals abroad, MRSA accounted for 60% to 80% of clinically isolated Staphylococcus aureus. In my country, from 1998 to 1999, the proportion of nosocomial MRSA accounted for more than 80% of Staphylococcus aureus, while the proportion of community-acquired MRSA accounted for nearly 22% of Staphylococcus aureus. The detection rates in various regions and hospitals have clearly increased year by year. Almost all MRSA are multidrug-resistant and are not sensitive to existing β-lactam antibiotics such as penicillins and cephalosporins, nor to chloramphenicol, clindamycin, aminoglycosides, tetracyclines, macrolide antibiotics, and quinolones. Glycopeptide antibiotics such as vancomycin have become the last line of defense for the treatment of MRSA infections.

The detection methods for methicillin-resistant Staphylococcus aureus currently used in clinical practice include: disk diffusion method (K-B method), broth (agar) dilution (MIC) method, concentration gradient (E-test) method, automated drug sensitivity testing, Real-Time PCR method and other detection methods.

The disc diffusion method (K-B method) is to add oxacillin to MH agar, adjust the bacterial solution to 0.5 McFarland turbidity, spread it on the above plate, incubate at 35℃ for 24h, the drug-resistant antibacterial graph is ≤11mm, and the sensitive antibacterial graph is ≥17mm. The biggest advantage of the K-B method is that it is fast, simple, cheap, and easy to be accepted by inspectors, but it is easy to cause missed detection. Affected by various factors in the culture environment, the drug resistance of some strains cannot be fully expressed.

The broth (agar) dilution (MIC) method is to add NaCl to 20g/L concentration in MH broth (or agar) culture medium, and at the same time add a certain amount of Ca and Mg ions, and dilute oxacillin in multiples to about 0.125-16μg/ml. The bacterial concentration is ¹⁰⁴ /mL, and incubate at 35℃ for 24h. MIC <2μg/mL means sensitive, and >4μg/mL means resistant. The detection rate of this method can reach 95%, and it has good repeatability, but the operation is more complicated than the instruction manual.

The concentration gradient (E-test) method is to paste the test strip of oxacillin on the MH agar plate containing 20g/L NaCl, adjust the bacterial solution to 0.5-1 McFarland turbidity, incubate at 35℃ for 24h, and read the MIC value directly. MIC value>4μg/mL is resistant, and MIC<2μg/mL is sensitive. The E-test method is more accurate in detecting low-level or moderately resistant MRSA. It is accurate, reliable, and stable, but expensive.

Automated drug sensitivity testing involves diluting the bacterial solution and injecting it into a drug sensitivity plate or well, and then interpreting the results by detecting the turbidity of the bacterial solution, the fluorescence intensity of the fluorescent indicator, or the hydrolysis reaction of the fluorescent substrate. Its advantage is that it is relatively fast, but sometimes it is difficult to achieve accurate detection levels within 3 to 4 hours for MRSA that grows slowly or expresses drug resistance late, and it is easy to miss or misreport MRSA. Currently, there are automated drug sensitivity testing products on the market, such as the Vitek system, ATB system, MicroScan system, and Sensiter ARIS.

Nucleic acid detection is mainly based on the Real-Time PCR method, which requires dozens of cycles of temperature changes, amplification reaction time is long, and the product is DNA, which is easy to be contaminated. Therefore, it is very necessary to develop a rapid, sensitive, specific and non-contaminating detection kit.

Simultaneous Amplification and Testing (SAT) is a method for direct and rapid detection of RNA. Compared with real-time fluorescent PCR for detecting DNA, the former detection system has an additional reverse transcription reaction step, and nucleic acid amplification is performed at a temperature (42°C) without the need for thermal cycling. M-MLV reverse transcriptase and T7 RNA polymerase are used for nucleic acid amplification. Compared with other nucleic acid amplification technologies, there are fewer reaction inhibitors and can effectively reduce false negative results. However, the problems faced by SAT technology in the detection of different types of viruses and bacteria are different, and specific analysis of the characteristics of viruses and bacteria is required for special design. At present, there are no reports on the research of real-time fluorescent nucleic acid isothermal amplification detection technology for methicillin-resistant Staphylococcus aureus (MRSA).

Summary of the invention

The purpose of the present invention is to provide a MRSA detection method which is rapid, highly accurate, easily contaminated, has simple equipment and is low in cost.

In a first aspect of the present invention, a method for amplifying methicillin-resistant Staphylococcus aureus (MRSA) is provided, the method comprising the steps of: performing amplification in a reaction system, the reaction system containing a specific primer pair for SA23s rRNA and mecA RNA of MRSA, the primer pair comprising:

(i) T7 primer for SA 23s rRNA: the sequence is shown in SEQ ID NO: 4;

(ii) nT7 primer for SA 23s rRNA: the sequence is shown in SEQ ID NO: 5;

(iii) T7 primer for mecA RNA: the sequence is shown in SEQ ID NO: 6; and

(iv) nT7 primer for mecA RNA: the sequence is shown in SEQ ID NO:7.

In another preferred embodiment, the reaction system also includes M-MLV reverse transcriptase and T7 RNA polymerase.

In another preferred embodiment, the reaction system further comprises: capture probes for methicillin-resistant Staphylococcus aureus MRSA SA 23srRNA and mecA RNA.

In another preferred embodiment, the reaction system further comprises: a specific detection probe for methicillin-resistant Staphylococcus aureus MRSA SA 23srRNA and a specific detection probe for mecA RNA.

In another preferred embodiment, the capture probes of MRSA SA 23s rRNA and mecA RNA can specifically bind to the target nucleic acid SA 23s rRNA and mecA RNA sequences of methicillin-resistant Staphylococcus aureus MRSA, respectively.

In another preferred example, the nucleotide sequence of the capture probe is shown in SEQ ID NO:3.

In another preferred embodiment, one end of the detection probe is labeled with a fluorescent group, and the other end is labeled with a quenching group.

In another preferred embodiment, the 5' end of the detection probe is labeled with a FAM fluorescent group, and the 3' end of the detection probe is labeled with a DABCYL quenching group.

In another preferred example, the nucleotide sequence of the MRSA SA 23s rRNA detection probe is shown in SEQ ID NO:8, and the sequence of the mecA RNA detection probe is shown in SEQ ID NO.:9.

In another preferred embodiment, the MRSA SA 23s rRNA detection probe and the mecA RNA detection probe are used to specifically bind to RNA copies generated by the DNA copies of the MRSA target nucleic acid SA 23s rRNA and mecA RNA respectively under the action of T7 RNA polymerase.

In another preferred embodiment, the method further comprises: performing an amplification reaction in another positive (control) reaction system or a negative (control) reaction system.

In another preferred embodiment, the control reaction system contains the specific primer pair, SA 23s rRNA internal standard and mecA RNA IC RNA internal standard, the capture probe and the detection probe.

In another preferred embodiment, the SA number of methicillin-resistant Staphylococcus aureus MRSA in the reaction system is less than 500 copies/ml, preferably 1-100 copies/ml, more preferably 1-50 copies/ml, and optimally 1-20 copies/ml; the mecA copy number is less than 1000 copies/ml, preferably 1-500 copies/ml, more preferably 1-100 copies/ml, and optimally 1-20 copies/ml.

In another preferred embodiment, the method further comprises the step of: during or after the amplification reaction, detecting the fluorescent signal emitted by the specific detection probe of methicillin-resistant Staphylococcus aureus MRSA SA 23s rRNA and mecA RNA.

In another preferred embodiment, the method is a real-time fluorescent nucleic acid isothermal amplification method.

In another preferred embodiment, the reaction system further contains the methicillin-resistant Staphylococcus aureus to be detected or a nucleic acid derived from SA 23s rRNA or mecA RNA of the methicillin-resistant Staphylococcus aureus.

In another preferred embodiment, the nucleic acid to be detected is a nucleic acid from a culture of methicillin-resistant Staphylococcus aureus.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In a second aspect of the present invention, a non-diagnostic detection method for methicillin-resistant Staphylococcus aureus (MRSA) is provided, the detection method comprising:

(a) providing a reaction system, wherein the reaction system contains a sample to be tested, and the reaction system also contains a specific detection probe for methicillin-resistant Staphylococcus aureus MRSA SA 23s rRNA and a specific detection probe for mecA RNA;

(b) amplifying the reaction system using the amplification method as described in the first aspect of the present invention; and

(c) During or after the amplification reaction, the fluorescence signals emitted by the specific detection probe for detecting MRSA SA 23srRNA and the specific detection probe for detecting mecA RNA.

In another preferred embodiment, the method further comprises setting up one or more control groups.

In another preferred embodiment, the control group includes: a MRSA positive control group, a MRSA negative control group, and a MRSA internal standard control group.

In a third aspect of the present invention, a detection kit for methicillin-resistant Staphylococcus aureus (MRSA) is provided, the kit comprising:

(a) a first container, and a specific primer pair for amplifying SA 23s rRNA of methicillin-resistant Staphylococcus aureus (MRSA) located in the container, wherein the primer pair comprises:

(i) T7 primer for SA 23s rRNA: the sequence is shown in SEQ ID NO: 4;

(ii) nT7 primer for SA 23s rRNA: the sequence is shown in SEQ ID NO: 5;

(b) a second container, and a specific primer pair for amplifying mecA RNA of methicillin-resistant Staphylococcus aureus (MRSA) located in the container, the primer pair comprising:

(iii) T7 primer for mecA RNA: the sequence is shown in SEQ ID NO: 6; and

(iv) nT7 primer for mecA RNA: the sequence is shown in SEQ ID NO:7;

and (c) instructions for use.

In another preferred embodiment, the first container and the second container are the same or different containers.

In another preferred embodiment, it further contains one or more components selected from the following group:

(d) capture probe;

(e) detection probe;

(f) MRSA internal standard sequence; and/or

(g) Internal standard detection probe.

In another preferred embodiment, it comprises one or more features selected from the following group:

The MRSA SA 23s rRNA detection probe comprises the nucleotide sequence shown in SEQ ID NO: 8, and the mecARNA detection probe comprises the nucleotide sequence shown in SEQ ID NO: 9; and/or

The capture probe comprises the nucleotide sequence shown in SEQ ID NO: 3; and/or

The MRSA SA 23s rRNA internal standard sequence comprises the nucleotide sequence shown in SEQ ID NO: 12, and the MRSA SA 23s rRNA internal standard detection probe comprises the nucleotide sequence shown in SEQ ID NO: 14; and/or

The mecA RNA internal standard sequence includes the nucleotide sequence shown in SEQ ID NO:13, and the mecA RNA internal standard detection probe includes the nucleotide sequence shown in SEQ ID NO:15.

In another preferred embodiment, the kit further comprises one or more enzymes.

In another preferred embodiment, the enzymes are M-MLV reverse transcriptase and T7 RNA polymerase.

In another preferred example, the M-MLV reverse transcriptase and T7 RNA polymerase are present in an enzyme solution, the capture probe is present in a nucleic acid extraction solution, the T7 primer, nT7 primer and detection probe of SA 23s rRNA are present in detection solution 1; the T7 primer, nT7 primer and detection probe of mecA RNA are present in detection solution 2.

In another preferred embodiment, the SA 23s rRNA internal standard detection probe is present in detection solution 1; and the mecA RNA internal standard detection probe is present in detection solution 2.

In another preferred embodiment, the MRSA internal standard is a competitive internal standard, and uses the same pair of primers (T7 and nT7 of SA 23s rRNA) as the MRSA target nucleotide (SA 23s rRNA); and uses the same pair of primers (T7 and nT7 of mecA RNA) as the MRSA target nucleotide (mecA RNA).

In another preferred embodiment, the kit further comprises a MRSA positive control and/or a MRSA negative control.

In another preferred embodiment, the kit comprises one or more of the following features:

The capture probe is a capture probe that specifically binds to the target nucleic acid SA 23s rRNA and mecARNA sequence of methicillin-resistant Staphylococcus aureus; and/or

The detection probe is an SA detection probe that specifically binds to an RNA copy generated from a DNA copy of the MRSA target nucleic acid SA 23s rRNA; and a mecA detection probe that specifically binds to an RNA copy generated from a DNA copy of the MRSA target nucleic acid mecA RNA.

In another preferred embodiment, the capture probe and/or the detection probe can also specifically bind to the target nucleic acid SA 23s rRNA and mecA RNA sequence of methicillin-resistant Staphylococcus aureus MRSA.

In another preferred embodiment, the kit comprises the following components: sample preservation solution, nucleic acid extraction solution, washing solution, amplification detection solution 1, expansion detection solution 2, enzyme solution, positive control, negative control and internal standard;

in,

The sample preservation solution includes ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) and HEPES;

The nucleic acid extraction solution includes capture probes and magnetic beads;

The washing solution comprises NaCl and SDS;

The amplification detection solution 1 includes dNTP and NTP, T7 primer of SA 23s rRNA, nT7 primer, detection probe and internal standard detection probe;

The amplification detection solution 2 includes dNTP and NTP, T7 primer of mecA RNA, nT7 primer, detection probe and internal standard detection probe;

The enzyme solution includes M-MLV reverse transcriptase and T7 RNA polymerase;

The positive control includes a mixture of in vitro transcribed RNA dilutions containing 10 ⁴ -10 ⁶ copies/mL of methicillin-resistant Staphylococcus aureus SA 23s rRNA and 10 ⁴ -10 ⁶ copies/mL of the drug-resistant mecA gene;

The negative control is a solution that does not contain the SA23s rRNA and mecA RNA sequence of methicillin-resistant Staphylococcus aureus or does not contain methicillin-resistant Staphylococcus aureus. Preferably, the negative control is physiological saline;

The internal standard is a mixture containing 10 ⁸ copies/mL of SA 23s rRNA IC RNA (SEQ ID No.: 12) dilution and 10 ⁵ copies/mL of mecA IC RNA (SEQ ID No.: 13) dilution.

In another preferred embodiment, the kit comprises box A and box B, wherein:

Box A is a specimen processing unit, comprising the sample preservation solution, the nucleic acid extraction solution and the washing solution;

Box B is a nucleic acid amplification detection unit, including the amplification detection solution 1, amplification detection solution 2, enzyme solution, positive control, negative control and internal standard.

In a fourth aspect of the present invention, a specific primer pair for amplifying methicillin-resistant Staphylococcus aureus MRSA 23srRNA and mecA RNA is provided, the primer pair comprising:

(i) T7 primer for SA 23s rRNA: the sequence is shown in SEQ ID NO: 4;

(ii) nT7 primer for SA 23s rRNA: the sequence is shown in SEQ ID NO: 5;

(iii) T7 primer for mecA RNA: the sequence is shown in SEQ ID NO: 6; and

(iv) nT7 primer for mecA RNA: the sequence is shown in SEQ ID NO:7.

In the fifth aspect of the present invention, there is provided a use of the kit as described in the third aspect of the present invention or the primer pair as described in the fourth aspect of the present invention for preparing a detection kit or a detection reagent for detecting methicillin-resistant Staphylococcus aureus (MRSA).

In another preferred embodiment, the detection kit or the detection reagent is used to detect whether methicillin-resistant Staphylococcus aureus (MRSA) exists in human nasal and pharyngeal swabs and wound secretions.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the fluorescence detection results of primer pairs and probe sequences of the SA alignment group;

Among them, the template concentrations of curves 1, 2, 3, 4 and 5 are 5×10 ⁵ copies/ml, 5×10 ⁴ copies/ml, 5000 copies/ml, 500 copies/ml and 50 copies/ml, respectively.

FIG2 shows the fluorescence detection results of the primer pairs and probe sequences of the SA present invention group;

Among them, the template concentrations of curves 1, 2, 3, 4 and 5 are 5×10 ⁵ copies/ml, 5×10 ⁴ copies/ml, 5000 copies/ml, 500 copies/ml and 50 copies/ml, respectively.

Figure 3 shows the fluorescence detection results of the primer pairs and probe sequences of the mecA alignment group;

Among them, the template concentrations of curves 1, 2, 3, 4 and 5 are 1×10 ⁶ copies/ml, 1×10 ⁵ copies/ml, 1×10 ⁴ copies/ml, 1000 copies/ml and 100 copies/ml, respectively.

FIG4 shows the fluorescence detection results of the primer pairs and probe sequences of the mecA present invention group;

Among them, the template concentrations of curves 1, 2, 3, 4 and 5 are 1×10 ⁶ copies/ml, 1×10 ⁵ copies/ml, 1×10 ⁴ copies/ml, 1000 copies/ml and 100 copies/ml, respectively.

FIG5 shows the SA target fluorescence detection results of SAT detection of cultured bacterial samples.

FIG6 is the SA internal standard fluorescence detection result of SAT detection of cultured bacterial samples.

FIG. 7 shows the fluorescence detection results of the mecA target by SAT detection of cultured bacterial samples.

FIG8 shows the mecA internal standard fluorescence detection results of SAT detection of cultured bacterial samples.

FIG. 9 shows the SA results of the cultured bacterial samples detected by using the methicillin-resistant Staphylococcus aureus nucleic acid detection kit (fluorescence PCR method).

FIG. 10 shows the mecA results of the cultured bacterial samples detected by using the methicillin-resistant Staphylococcus aureus nucleic acid detection kit (fluorescence PCR method).

Detailed ways

After long-term and in-depth research, the inventors have developed for the first time a highly specific and sensitive primer pair for detecting methicillin-resistant Staphylococcus aureus (MRSA) and a matching specific detection probe after a large number of screening and verification of target sequences, primers and probes. Specifically, when the two specific nucleic acid targets (SA 23s rRNA and mecA RNA) of methicillin-resistant Staphylococcus aureus (MRSA) are amplified using this specific primer sequence, it has excellent advantages such as high specificity and high sensitivity, and can be used to accurately detect methicillin-resistant Staphylococcus aureus (MRSA) and distinguish other Staphylococci. The present invention was completed on this basis.

Primers and probes

As used herein, the term "two pairs of specific primers (SA 23srRNA and mecA RNA) for methicillin-resistant Staphylococcus aureus (MRSA)" refers to two sets of primers (pairs) that amplify products having the 23s rRNA chain of SA and the mRNA chain of mecA gene of methicillin-resistant Staphylococcus aureus (MRSA).

In primer design, in order to better meet the current needs for the detection of methicillin-resistant Staphylococcus aureus (MRSA), the inventors selected highly conserved and highly specific segments in the 23s rRNA and drug-resistant gene mecA of methicillin-resistant Staphylococcus aureus as amplification target sequence regions (the nucleotide sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2). Preferred primer sequences include the SA 23s rRNA primer pair composed of SEQ ID NO: 4 and SEQ ID NO: 5 and the mecA RNA primer pair composed of SEQ ID NO: 6 and SEQ ID NO: 7.

In the amplification method of the present invention, in addition to primers, the reaction system may also include one or more probes, such as detection probes, capture probes, and the like.

As used herein, the term "capture probe for methicillin-resistant Staphylococcus aureus (MRSA)" refers to a nucleotide sequence that can specifically bind to the target nucleic acid (SA 23s rRNA and mecA gene mRNA) sequence of methicillin-resistant Staphylococcus aureus (MRSA). A preferred capture probe of the present invention has a nucleotide sequence as shown in SEQ ID NO:3. More preferably, the capture probe specifically binds to the target nucleic acid SA 23s rRNA and mecA gene mRNA sequence of methicillin-resistant Staphylococcus aureus (MRSA). In another preferred embodiment, when the methicillin-resistant Staphylococcus aureus (MRSA) contains MRSA internal standards (SA and mecA gene IC RNA), the capture probe can also specifically bind to the MRSA internal standard sequence (SA and mecA gene IC RNA) for designing a negative control group.

MRSA detection probes are all molecular beacons, which are a class of highly specific and highly sensitive molecular probes, composed of single-stranded nucleic acid molecules covalently labeled with fluorescent dyes and quenchers at both ends, and present a hairpin type or stem-loop structure. The loop part of the molecular beacon is complementary to the target, and the two ends become stems due to complementarity. Compared with the linear TaqMan probe, the molecular beacon probe requires a certain force to open its hairpin structure, so its specificity is better than that of the linear probe. The two preferred detection probes for SA23s rRNA and mecA gene mRNA of the present invention have nucleotide sequences shown in SEQ ID NO:8 and SEQ ID NO:9, respectively.

The primer sequences and probe sequences of the invention are designed according to the primer and probe design principle, using DNAStar, DNAMAN software and artificial design to be special primer and probe sequences for real-time fluorescent nucleic acid isothermal amplification to detect methicillin-resistant Staphylococcus aureus (MRSA).

For the present invention, the preferred primer pairs and probes are as follows:

Control

Since SAT amplification is easily affected by various factors, which may lead to amplification failure and cause the kit user to make wrong judgments and draw erroneous conclusions, a control substance may be provided in the kit of the present invention to eliminate the situation where the test results are distorted.

The reference objects that can be provided in the present invention include: a MRSA positive control, a MRSA negative control, and one or more of the MRSA internal standards.

The MRSA positive control can be an in vitro transcribed RNA that has no biological activity. By testing the positive control, it can be proved that the test method and materials of the kit are correct, ensuring the accuracy of the test, and at the same time monitoring the repeatability and stability of each test and the differences between kit batches.

In addition, a critical weak-positive control can be prepared using a positive control (physiological saline and lysis buffer are mixed in a 1:1 ratio to form a diluent, and both SA 23s rRNA and mecA genes are diluted 100 times as the positive control as a critical weak-positive control), which can indicate the test operation status when it is in a critical value state. By regularly testing the SAT laboratory through the critical weak-positive control, indoor quality control can be performed to prevent missed detection (false negatives) during the test process.

The MRSA internal standard can be an in vitro transcribed RNA that has no biological activity. The MRSA internal standard is used as a competitive internal standard for MRSA (SA23s rRNA and mecA gene) RNA, and can be used as a reference to prevent the occurrence of false negative results. By testing the sample with the internal standard added, it can be understood whether the entire amplification reaction system is inhibited, which can better indicate false negatives.

Negative controls can exclude false positives and ensure the specificity of the test when the kit detection methods and materials are used correctly.

In another preferred embodiment, the in vitro transcribed MRSA (SA23srRNA and mecA RNA) is prepared by the following method:

(1) synthesizing the SA 23s rRNA and drug-resistant mecA gene fragments of MRSA by chemical synthesis (the nucleotide sequences of the SA 23s rRNA and drug-resistant mecA positive fragments are shown in sequences 10 and 11 in the sequence table);

(2) Insert the SA 23s rRNA and drug-resistant mecA gene fragments into -T vector, construct the positive control plasmids of SA 23srRNA and drug-resistant mecA;

(3) The positive control plasmids of SA 23s rRNA and drug-resistant mecA were transformed into Escherichia coli DH5α and named -T-SA23s rRNA and/> -T-mecA strain, stored at -70°C;

(4) respectively -T-SA23s rRNA and/> -T-mecA strain extracted/> -T-SA23srRNA and/> -T-mecA plasmid, transcribe RNA from the plasmid, purify to remove DNA, and quantify and identify RNA.

In another preferred embodiment, the in vitro transcribed MRSA (SA 23s rRNA and drug-resistant mecA gene) IC RNA in the MRSA internal standard is prepared by the following method:

(1) synthesizing by chemical synthesis a segment whose sequences are basically the same as the SA 23s rRNA and drug-resistant mecA target sequence regions of MRSA except for the probe detection region sequence (the nucleotide sequences of the SA 23s rRNA and drug-resistant mecA internal standard fragments of MRSA are shown in SEQ ID No.: 12 and 13 in the sequence list, respectively);

(2) Clone the SA 23s rRNA and drug-resistant mecA gene fragments into -T vector, construct the internal standard plasmids of MRSA SA23s rRNA and drug-resistant mecA;

(3) MRSA SA 23s rRNA and drug-resistant mecA internal standard plasmid were transformed into Escherichia coli DH5α and named -T-SA23s rRNA IC and/> -T-mecA IC strain, stored at -70°C;

(4) respectively -T-SA23s rRNA IC and/> -T-mecA IC strain extracted/> -T-SA23s rRNA IC and/> -T-mecA IC plasmid, transcribe the plasmid RNA to purify the DNA, and quantify and identify the internal standard RNA.

Detection method

The present invention also provides a method for detecting methicillin-resistant Staphylococcus aureus (MRSA), comprising amplifying SA 23s rRNA and drug-resistant mecA of a sample to be tested using the specific primers of the present invention; and during or after the amplification reaction, respectively detecting the SA 23s rRNA and drug-resistant mecA amplification products of methicillin-resistant Staphylococcus aureus (MRSA), for example, by detecting the fluorescent signal emitted by the specific probe.

The method may also optionally include setting up two or more control groups, such as a MRSA positive control group, a MRSA negative control group, a MRSA internal standard group, and the like.

In the present invention, the detection method can be a conventional PCR method, or a different method such as a real-time fluorescence detection method. A particularly preferred method is a real-time fluorescence nucleic acid isothermal amplification method.

Real-time fluorescence nucleic acid isothermal amplification technology (SAT)

Nucleic acid constant temperature amplification real-time fluorescence detection technology is a new type of nucleic acid detection technology that combines RNA constant temperature amplification and real-time fluorescence detection. First, it is achieved simultaneously through M-MLV reverse transcriptase, T7 RNA polymerase and optimized probe (optimal probe, patent pending) technology. Reverse transcriptase is used to produce a DNA copy of the target nucleic acid (RNA), T7 RNA polymerase produces multiple RNA copies from the DNA copy, and the optimized probe with fluorescent label specifically binds to these RNA copies to generate fluorescence, which can be captured by the detection instrument. The test results are judged based on the appearance time and intensity of the real-time fluorescence signal, combined with the positive control, negative control and internal standard signal.

In the present invention, the universal detection technology for methicillin-resistant Staphylococcus aureus (MRSA) can efficiently and specifically capture the SA 23s rRNA and drug-resistant mecA RNA of MRSA by using a dedicated primer pair with high specificity and high sensitivity for two groups of targets and using a dedicated capture probe. Nucleic acid amplification is achieved simultaneously using M-MLV reverse transcriptase and T7 RNA polymerase, the reverse transcriptase is used to produce a DNA copy of the target nucleic acid RNA, and the T7 RNA polymerase produces multiple RNA copies from the DNA copy. The optimized detection probe with a fluorescent label specifically binds to the RNA copy produced after amplification, thereby generating fluorescence, and the fluorescent signal can be captured by a detection instrument.

Reagent test kit

The present invention provides a detection kit for methicillin-resistant Staphylococcus aureus (MRSA), comprising:

(a) a specific primer pair for amplifying SA 23s rRNA and drug-resistant mecA, wherein the amplified product amplified by the primers has MRSA SA 23s rRNA and drug-resistant mecA mRNA sequences;

(b) capture probe;

(c) Detection probe.

The primer pair is a pair of SA 23s rRNA and drug-resistant mecA amplification primers used to produce DNA copies of MRSA SA 23s rRNA and drug-resistant mecA target nucleic acid (SA 23s rRNA and drug-resistant mecA RNA) under the action of M-MLV reverse transcriptase. In a preferred example of the present invention, the MRSA amplification primers include: SA 23s rRNA T7 primer: SEQ ID NO: 4; and nT7 primer: SEQ ID NO: 5; drug-resistant mecA T7 primer: SEQ ID NO: 6; and nT7 primer: SEQ ID NO: 7.

The capture probe is a capture probe that specifically binds to the target nucleic acid (SA 23s rRNA and drug-resistant mecA RNA) sequence of methicillin-resistant Staphylococcus aureus (SA 23s rRNA and drug-resistant mecA RNA); the detection probe is a MRSA SA 23s rRNA and drug-resistant mecA RNA detection probe that specifically binds to RNA copies generated from DNA copies of the MRSA SA 23s rRNA and drug-resistant mecA target nucleic acid (SA 23s rRNA and drug-resistant mecA RNA). In another preferred embodiment, the capture probe and/or the detection probe can also specifically bind to the target nucleic acid (SA 23s rRNA and drug-resistant mecA RNA) sequence of methicillin-resistant Staphylococcus aureus (MRSA).

The methicillin-resistant Staphylococcus aureus (MRSA) can also be designed to contain MRSA internal standards (SA 23srRNA IC and drug-resistant mecA IC RNA). When the methicillin-resistant Staphylococcus aureus contains MRSA (SA 23s rRNA and drug-resistant mecA RNA) internal standards, the capture probe and the detection probe are designed to specifically bind to the MRSA (SA 23srRNA and drug-resistant mecA RNA) internal standard sequences, respectively, to form MRSA (SA 23s rRNA and drug-resistant mecA RNA) internal standard control groups. In another preferred embodiment, the MRSA (SA 23s rRNA and drug-resistant mecA RNA) internal standards are all competitive internal standards, and use the same pair of primers (T7 and nT7) with the MRSA (SA 23s rRNA and drug-resistant mecA RNA) target nucleotides (SA 23s rRNA and drug-resistant mecA RNA).

The kit may also include one or more enzymes, such as M-MLV reverse transcriptase and T7 RNA polymerase. In another preferred embodiment, the M-MLV reverse transcriptase and T7 RNA polymerase are present in an enzyme solution, the capture probe is present in a nucleic acid extraction solution, the SA T7 primer, nT7 primer and detection probe are present in the same detection solution, and the mecA T7 primer, nT7 primer and detection probe are separately present in the same detection solution.

In another preferred embodiment, the kit comprises the following components: sample preservation solution, nucleic acid extraction solution, washing solution, SA23s rRNA amplification detection solution, mecA amplification detection solution, enzyme solution, MRSA positive control, MRSA negative control and MRSA internal standard; wherein the lysis solution comprises ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) and HEPES;

The nucleic acid extraction solution includes capture probes and magnetic beads;

The washing solution comprises NaCl and SDS;

The SA 23s rRNA amplification detection solution includes dNTP and NTP; SA T7 primer, nT7 primer, SA detection probe and internal standard detection probe;

The mecA amplification detection solution includes dNTP and NTP; mecA T7 primer, nT7 primer, mecA detection probe and internal standard detection probe;

The enzyme solution includes M-MLV reverse transcriptase and T7 RNA polymerase;

The MRSA positive controls included SA 23s rRNA of methicillin-resistant Staphylococcus aureus and in vitro transcribed RNA dilutions of the drug-resistant mecA gene;

The MRSA negative control is a methicillin-resistant Staphylococcus aureus target nucleic acid (SA 23s rRNA and drug-resistant mecARNA) sequence or a solution that does not contain methicillin-resistant Staphylococcus aureus. Preferably, the negative control is physiological saline.

MRSA internal standard: contains dilutions of MRSA internal standard RNA (SA 23s rRNA IC RNA and drug-resistant mecA RNA, the sequences are shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively).

In another preferred embodiment, the various reagents in one reaction unit of the kit are composed as follows:

(1) Sample storage solution: HEPES 25-250mM, (NH ₄ ) ₂ SO ₄ 5-50mM;

(2) Nucleic acid extraction solution: HEPES 50-400 mM, EDTA 40-200 mM, LiCl 400-2000 mM, capture probe 1-50 μM (preferably 5-25 μM), magnetic beads 50-500 mg/L (preferably 50-250 mg/L);

(3) Washing solution: HEPES 5-50 mM, NaCl 50-500 mM, 1% SDS, EDTA 1-10 mM;

(4) SA 23s rRNA amplification detection solution: prepare a reaction solution with Tris 10-50mM, MgCl2 10-40mM, dNTP 0.1-10mM (preferably 0.5-5mM), NTP 1-20mM (preferably 1-10mM), PVP40 1-10%, and KCl 5-40mM; prepare a SA 23s rRNA amplification primer and a SA 23s rRNA detection probe by dissolving them in TE solution (a mixture of 10mM Tris and 1mM EDTA), wherein the concentration of each primer and probe can be 5-10pmol/reaction; wherein the concentration of T7 primer is preferably 7.5pmol/reaction, the concentration of nT7 primer is preferably 7.5pmol/reaction, the concentration of SA 23s rRNA detection probe is preferably 5pmol/reaction, and the concentration of internal standard detection probe is preferably 5pmol/reaction to prepare the detection solution; and mix the two in a ratio of 16:1.

(5) mecA amplification detection solution: Tris 10-50mM, MgCl2 10-40mM, dNTP 0.1-10mM (preferably 0.5-5mM), NTP 1-20mM (preferably 1-10mM), PVP40 1-10%, KCl 5-40Mm are prepared as a reaction solution; mecA amplification primers and mecA detection probes are dissolved in TE solution (a mixture of 10mM Tris and 1mM EDTA) to prepare the solution, and the concentration of each primer and probe can be 5-10pmol/reaction; wherein the concentration of T7 primers is preferably 5pmol/reaction, the concentration of nT7 primers is preferably 2.5pmol/reaction, the concentration of mecA detection probes is preferably 5pmol/reaction, and the concentration of internal standard detection probes is preferably 5pmol/reaction to prepare the detection solution; the two are mixed at a ratio of 16:1.

(6) Enzyme solution: M-MLV reverse transcriptase 400-4000 U/reaction (preferably 500-1500 U/reaction), T7 RNA polymerase 200-2000 U/reaction (preferably 500-1000 U/reaction), 2-10 mM HEPES pH 7.5, 10-100 mM N-acetyl-L-cysteine, 0.04-0.4 mM zinc acetate, 10-100 mM trehalose, 40-200 mM Tris-HCl pH 8.0, 40-200 mM KCl, 0.01-0.5 mM EDTA, 0.1-1% (v/v) Triton X-100 and 20-50% (v/v) glycerol;

(7) MRSA positive control: a mixture containing 10 ⁴ -10 ⁶ copies/mL of SA 23s rRNA and 10 ⁴ -10 ⁶ copies/mL of in vitro transcribed RNA dilutions of the drug-resistant mecA gene;

(8) MRSA negative control: a solution that does not contain the target nucleic acid (SA 23s rRNA and mecA RNA) sequence of methicillin-resistant Staphylococcus aureus or does not contain methicillin-resistant Staphylococcus aureus;

(9) MRSA internal standard: a mixture containing 10 ⁸ copies/mL of SA 23s rRNA IC RNA (sequence as shown in Sequence 12 in the sequence listing) dilution and 10 ⁵ copies/mL of mecA IC RNA (sequence as shown in Sequence 13 in the sequence listing) dilution.

In another preferred embodiment, the kit further comprises one or more containers, and the above-mentioned components can be respectively located in one or more containers. In another preferred embodiment, the kit comprises box A and box B, wherein:

Box A is a specimen processing unit, including the sample storage, the nucleic acid extraction solution and the washing solution;

Box B is a nucleic acid amplification detection unit, including the SA 23s rRNA amplification detection solution and mecA amplification detection solution, enzyme solution, MRSA positive control, MRSA negative control and MRSA internal standard.

In another preferred example, the detection probe comprises the nucleotide sequence shown as SEQ ID NO:8, SEQ ID NO:9, and/or the capture probe comprises the nucleotide sequence shown as SEQ ID NO:3.

The above kit is used to perform real-time fluorescent nucleic acid isothermal amplification detection on methicillin-resistant Staphylococcus aureus, including the following steps:

1) Lysing the methicillin-resistant Staphylococcus aureus in the sample to be tested with a sample preservation solution to obtain a lysate containing methicillin-resistant Staphylococcus aureus nucleic acid;

2) adding a nucleic acid extraction solution and MRSA IC RNA to the lysate of step 1), allowing the capture probe to specifically bind to the target or internal standard nucleic acid and then to bind to the magnetic beads, washing with a washing solution to remove the nucleic acid not bound to the magnetic beads, and obtaining nucleic acid (SA 23s rRNA and mecA RNA) of methicillin-resistant Staphylococcus aureus and MRSA IC RNA (SA 23s rRNA ICRNA and mecA IC RNA);

3) adding the nucleic acid (SA 23s rRNA and mecA RNA) of methicillin-resistant Staphylococcus aureus and MRSA IC RNA (SA 23s rRNA IC RNA and mecA IC RNA) extracted in step 2) to the first-stage reactant consisting of SA 23s rRNA amplification detection solution and mecA amplification detection solution, incubating at 60° C. for 10 minutes, and then incubating at 42° C. for 5 minutes, and then adding the second-stage enzyme reactant enzyme solution, and then continuing to incubate at 42° C. for 60 minutes, and synchronously recording the change of the fluorescence signal with a detector; the volume ratio of the first-stage reactant to the second-stage enzyme reactant is 3:1;

4) According to the time and intensity of the fluorescence signal generation, the SA 23s rRNA and mecA samples were comprehensively qualitatively tested with reference to the MRSA positive control, MRSA negative control and MRSA internal standard test results.

In the above detection operation, the sample to be tested in step 1) is a culture of methicillin-resistant Staphylococcus aureus.

Compared with the existing methicillin-resistant Staphylococcus aureus detection, the present invention has the following advantages:

(1) High specificity, high purity, and low contamination: The optimal capture probe designed for MRSA target nucleic acid (SA 23s rRNA and mecA) can efficiently and specifically capture MRSA (SA 23s rRNA and mecA) RNA. At the same time, due to the use of a closed constant temperature amplification detection system, the reaction system does not need to be opened during the entire reaction process, thus avoiding the contamination of the amplicon.

(2) Rapid detection: Nucleic acid amplification and detection are carried out simultaneously in the same closed system, and there is no temperature increase or decrease or circulation during the entire process, so the time required is greatly shortened. Amplification detection only takes 40 minutes.

(3) Contamination is easy to control: Compared with real-time fluorescence PCR, the amplification product of the present invention is RNA, which is easily degraded in nature, so contamination control is easier.

(4) Simple equipment and low cost: Compared with real-time fluorescence quantitative PCR, the instrument used in the present invention does not require temperature rise and fall cycles, thereby greatly reducing the design and production costs.

In summary, the kit of the present invention can detect RNA of methicillin-resistant Staphylococcus aureus in nasal and pharyngeal swabs, wound secretions or sterile body fluids such as cerebrospinal fluid, and has the characteristics of high specificity, high sensitivity (up to 50 copies/mL and 100 copies/mL), low contamination (amplified product RNA is easily degraded in the natural environment) and rapid detection (amplification detection is completed in 40 minutes). It will play an important role in the clinical diagnosis of early methicillin-resistant Staphylococcus aureus infection and has broad application prospects.

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

The main raw materials, enzyme solution, positive control and internal standard in vitro transcribed RNA used in the examples were provided by RDBiosciences, USA. The 7500 PCR instrument was a product of ABI, USA. Reagents such as NTPs, dNTPs and other instruments were all conventional commercially available products.

Example 1 Design of special primers and probes for real-time fluorescent nucleic acid isothermal amplification to detect methicillin-resistant Staphylococcus aureus (MRSA)

The present invention selects a highly conserved and highly specific segment in the MRSA (SA 23s rRNA and mecA) gene as the amplification target sequence region (the nucleotide sequence thereof is shown in SEQ ID No.: 1 and SEQ ID No.: 2), and according to the primer and probe design principle, uses DNAStar, DNAMAN software and artificially designed special primers and probe sequences for real-time fluorescent nucleic acid isothermal amplification detection of methicillin-resistant Staphylococcus aureus (SA 23s rRNA and mecA of MRSA), and obtains the following specific sequence:

(1) a capture probe (TCO, Target Capture Oligo) that can specifically bind to a target nucleic acid (SA 23s rRNA) sequence of methicillin-resistant Staphylococcus aureus (MRSA), wherein the nucleotide sequence of the capture probe is: 5'TGCACACCGGATGGCCAATCCAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3' (SEQ ID No.: 3); a capture probe (TCO, Target Capture Oligo) that can specifically bind to a target nucleic acid (mecA RNA) sequence of methicillin-resistant Staphylococcus aureus (MRSA), wherein the capture probes are identical, and the nucleotide sequence thereof is: 5'TGCACACCGGATGGCCAATCCAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3' (SEQ ID No.: 3);

(2) a pair of SA amplification primers for producing DNA copies of the MRSA target nucleic acid SA 23s rRNA and a pair of mecA amplification primers for producing DNA copies of the target nucleic acid mecA under the action of M-MLV reverse transcriptase, wherein the SA 23s rRNA and mecA amplification primers are composed of a single T7 primer and an nT7 primer, respectively, and the SA T7 primer sequence is 5'AATTTAATACGACTCACTATAGGGAGACATATTTC

TCTACACCTTT 3' (SEQ ID No.: 4), and the nT7 primer sequence is 5'AGCGATTGATGGTGATACGGTT 3' (SEQ ID No.: 5);

The mecA T7 primer sequence is 5'AATTTAATACGACTCACTATAGGGAGATTCTTTAGCGATTGCTTTATAATC 3' (SEQ ID No.: 6), and the nT7 primer sequence is 5'AATCAGAACGTGGTAAAA 3' (SEQ ID No.: 7);

(3) an SA detection probe for specifically binding to an RNA copy generated by a DNA copy of the MRSA target nucleic acid SA 23s rRNA under the action of T7 RNA polymerase, the nucleotide sequence of the SA detection probe being 5'CCGUCAUUCAGACUAUUAUUGGACGG 3' (SEQ ID No.: 8), the 5' end being fluorescently labeled with FAM, and the 3' end being fluorescently labeled with DABCYL; a mecA detection probe for specifically binding to an RNA copy generated by a DNA copy of the MRSA target nucleic acid mecA RNA under the action of T7 RNA polymerase, the nucleotide sequence of the mecA detection probe being 5'CACAUCAGGAACAGCAUAUGAGAUGUG 3' (SEQ ID No.: 9), the 5' end being fluorescently labeled with FAM, and the 3' end being fluorescently labeled with DABCYL.

(4) Other comparison sequences

In the process of primer and probe design, many pairs of primer and probe sequences are generated, such as dozens of pairs of primers and corresponding detection probes. For example, for the target SA, the following primer pairs and detection probes are included:

T7 primer S1a: 5'AATTTAATACGACTCACTATAGGGAGACCGTATCACCATCAATCGCTT 3' (as shown in SEQ ID NO: 18),

nT7 primer S1b: 5'CAAATGCATCACAAACAGA 3' (as shown in SEQ ID NO: 19),

The detection probe S1c is 5'CGCUCUGAAGAUCCAACAGUGAGCG 3' (as shown in SEQ ID NO: 20),

The 5' end of the detection probe is fluorescently labeled with FAM, and the 3' end is fluorescently labeled with DABCYL.

For the target MecA, the following primer pairs and detection probes were included:

T7 primer M1a: 5'AATTTAATACGACTCACTATAGGGAGAATGCTTTGGTCTTTCTG 3' (as shown in SEQ ID NO: 21),

nT7 primer M1b: 5'CTACGGTAACATTGATCGCA 3' (as shown in SEQ ID NO: 22),

Detection probe M1c 5'CGCCGGUGGAAGUUAGAUUGGGCGGCG 3' (as shown in SEQ ID NO: 23),

The 5' end of the detection probe is fluorescently labeled with FAM, and the 3' end is fluorescently labeled with DABCYL.

(5) Amplification screening

For each set of designed sequences, the same reaction system is used for amplification, sensitivity is compared and the better one is selected. The best primer and probe sequences with high detection sensitivity are selected by screening and comparing each designed primer pair and probe sequence.

Some of the results are shown in Figures 1-4.

The results of SA are shown in Figures 1 and 2 (Figure 1 is the comparison group, and Figure 2 is the present invention group); the results of mecA are shown in Figures 3 and 4 (Figure 3 is the comparison group, and Figure 4 is the present invention group).

The sensitivity of the SA comparison group (using S1a, S1b primer pair and S1c detection probe) can detect 500 copies/mL, while the sensitivity of the present invention group is the best, reaching 50 copies/mL. The sensitivity of the present invention group is at least improved by one order of magnitude.

The sensitivity of the mecA comparison group (using M1a, M1b primer pair and M1c detection probe) can detect 1000 copies/mL, while the sensitivity of the present invention group is the best, reaching 100 copies/mL. The sensitivity of the present invention group is at least improved by one order of magnitude, reaching 100 copies/mL or lower.

In addition, it can be clearly seen from the shape of the curve that both inventive groups are superior to the control group.

Therefore, the preferred primer pairs were determined as follows: for SA, the primer pair was SEQ ID NO: 4, SEQ ID NO: 5; the probe sequence was SEQ ID NO: 8;

For mecA, the primer pair is SEQ ID NO: 6, SEQ ID NO: 7; and the probe sequence is SEQ ID NO: 9.

(6) Screening of internal standards and internal standard detection probes

To facilitate result analysis, the MRSA internal standard (SEQ ID No.: 12; SEQ ID No.: 13) added to the kit is also used to optimize the internal standard detection probes. The MRSA (SA 23s rRNA and mecA) internal standard and the MRSA target nucleotide ((SA 23s rRNA and mecA) RNA) have the same primer binding region, and the nucleic acid sequence or arrangement between the two primers is different, so that they cannot bind to the detection probe, but can bind to the internal standard probe. The MRSA internal standard can be obtained by site-directed mutagenesis of the MRSA (SA 23s rRNA and mecA) target template, and can specifically bind to the capture probe. The internal standard detection probe is a probe with a different sequence and fluorescent label from the MRSA (SA 23s rRNA and mecA) detection probe. The nucleotide sequence of the internal standard detection probe is SA: 5'CCGACAGUGAUACGAGAGTCGG 3' (SEQ ID No.: 14), mecA: 5'CCGACCCAGGUAUCAGUGTCGG3' (SEQ ID No.:15), the 5' end is labeled with a HEX fluorescent group, and the 3' end is labeled with a DABCYL quenching group.

Example 2 Preparation of a real-time fluorescent nucleic acid isothermal amplification detection kit for methicillin-resistant Staphylococcus aureus (MRSA)

The real-time fluorescent nucleic acid isothermal amplification detection kit for methicillin-resistant Staphylococcus aureus (MRSA) of the present invention is obtained by using the special primers and probes provided in Example 1. The kit comprises components such as capture probe (TCO, Target Capture Oligo), T7 primer, nT7 primer, MRSA (SA 23srRNA) detection probe, SA internal standard detection probe, MRSA (mecA) detection probe, mecA internal standard detection probe, internal standard, M-MLV reverse transcriptase and T7 RNA polymerase.

The capture probe is present in the nucleic acid extract, the T7 primer, nT7 primer, MRSA detection probe, and internal standard detection probe are respectively present in the MRSA (SA 23srRNA) detection solution and the MRSA (mecA) detection solution, and the M-MLV reverse transcriptase and T7 RNA polymerase are present in the enzyme solution. Specifically, the kit is divided into a box A (specimen processing unit) stored at 2-30°C and a box B (nucleic acid amplification detection unit) stored at -15--35°C. The box A includes a sample preservation solution, a nucleic acid extract, and a washing solution, and the box B includes an amplification detection solution 1, an amplification detection solution 1, an enzyme solution, a MRSA positive control, a MRSA negative control, and a MRSA internal standard. The main components are as follows:

Box A (specimen processing unit) consists of:

Sample preservation solution; contains ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) and HEPES;

Nucleic acid extract: containing capture probe 1-50 μM (preferably 5-25 μM) and magnetic beads 50-500 mg/L (preferably 50-250 mg/L);

Washing solution: mainly contains 1% (V/V) SDS.

The B box (nucleic acid amplification detection unit) consists of:

Amplification detection solution 1: containing dNTP 0.1-10mM (preferably 0.5-5mM), NTP 1-20mM (preferably 1-10mM); the concentration of each SA primer and probe can be 5-10pmol/reaction, wherein the concentration of T7 primer is preferably 7.5pmol/reaction, the concentration of nT7 primer is preferably 7.5pmol/reaction, the concentration of SA detection probe is preferably 5pmol/reaction, and the concentration of internal standard detection probe is preferably 5pmol/reaction;

Amplification detection solution 2: containing dNTP 0.1-10mM (preferably 0.5-5mM), NTP 1-20mM (preferably 1-10mM); the concentration of each mecA primer and probe can be 5-10pmol/reaction, wherein the concentration of T7 primer is preferably 2.5pmol/reaction, the concentration of nT7 primer is preferably 5pmol/reaction, the concentration of mecA detection probe is preferably 5pmol/reaction, and the concentration of internal standard detection probe is preferably 5pmol/reaction;

Enzyme solution: containing M-MLV reverse transcriptase 400-4000U/reaction (preferably 500-1500U/reaction), T7 RNA polymerase 200-2000U/reaction (preferably 500-1000U/reaction);

MRSA positive control; a mixture containing 10 ⁴ -10 ⁶ copies/mL of SA 23s rRNA and 10 ⁴ -10 ⁶ copies/mL of in vitro transcribed RNA dilutions of the drug-resistant mecA gene;

MRSA negative control: a solution without the target nucleic acid (SA 23s rRNA and mecA RNA) sequence of methicillin-resistant Staphylococcus aureus or without methicillin-resistant Staphylococcus aureus;

MRSA internal standard: a mixture containing 10 ⁸ copies/mL of SA 23s rRNA IC RNA (SEQ ID No.: 12) dilution and 10 ⁵ copies/mL of mecA IC RNA (SEQ ID No.: 13) dilution.

All reagents contained in the kit can be prepared according to conventional methods or purchased commercially.

Specifically, in each reaction unit, the specific combination of the various reagents in the kit is as follows:

(1) Sample storage solution: HEPES 25-250mM, (NH ₄ ) ₂ SO ₄ 5-50mM;

(2) Nucleic acid extraction solution: HEPES 50-400 mM, EDTA 40-200 mM, LiCl 400-2000 mM, capture probe 1-50 μM (preferably 5-25 μM), magnetic beads 50-500 mg/L (preferably 50-250 mg/L);

(3) Washing solution: HEPES 5-50 mM, NaCl 50-500 mM, 1% SDS, EDTA 1-10 mM;

(4) Amplification detection solution 1: containing dNTP 0.1-10mM (preferably 0.5-5mM), NTP 1-20mM (preferably 1-10mM); the concentration of each SA primer and probe can be 5-10pmol/reaction, wherein the concentration of T7 primer is preferably 7.5pmol/reaction, the concentration of nT7 primer is preferably 7.5pmol/reaction, the concentration of SA detection probe is preferably 5pmol/reaction, and the concentration of internal standard detection probe is preferably 5pmol/reaction;

(5) Amplification detection solution 2: containing dNTP 0.1-10 mM (preferably 0.5-5 mM), NTP 1-20 mM (preferably 1-10 mM); the concentration of each mecA primer and probe can be 5-10 pmol/reaction, wherein the concentration of T7 primer is preferably 2.5 pmol/reaction, the concentration of nT7 primer is preferably 5 pmol/reaction, the concentration of mecA detection probe is preferably 5 pmol/reaction, and the concentration of internal standard detection probe is preferably 5 pmol/reaction;

(6) Enzyme solution: containing 400-4000 U/reaction (preferably 500-1500 U/reaction) of M-MLV reverse transcriptase and 200-2000 U/reaction (preferably 500-1000 U/reaction) of T7 RNA polymerase;

(7) MRSA positive control: a mixture containing 10 ⁴ -10 ⁶ copies/mL of SA 23s rRNA and 10 ⁴ -10 ⁶ copies/mL of in vitro transcribed RNA dilutions of the drug-resistant mecA gene;

(8) MRSA negative control: a solution that does not contain the target nucleic acid (SA 23s rRNA and mecA RNA) sequence of methicillin-resistant Staphylococcus aureus or does not contain methicillin-resistant Staphylococcus aureus;

(9) MRSA internal standard: a mixture containing 10 ⁸ copies/mL of SA 23s rRNA IC RNA (SEQ ID No.: 12) dilution and 10 ⁵ copies/mL of mecA IC RNA (SEQ ID No.: 13) dilution.

The in vitro transcribed MRSA (SA23srRNA and mecA RNA) in the MRSA positive control can be prepared by a variety of methods, one of which is as follows:

(1) synthesizing the SA 23s rRNA and drug-resistant mecA gene fragments of MRSA by chemical synthesis (SA 23s rRNA and drug-resistant mecA positive fragments, whose nucleotide sequences are shown in SEQ ID No.: 10 and SEQ ID No.: 11 respectively);

(2) Insert the SA 23s rRNA and drug-resistant mecA gene fragments into -T vector, construct the positive control plasmids of SA 23srRNA and drug-resistant mecA;

(3) The positive control plasmids of SA 23s rRNA and drug-resistant mecA were transformed into Escherichia coli DH5α and named -T-SA23s rRNA and/> -T-mecA strain, stored at -70°C;

(4) respectively -T-SA23s rRNA and/> -T-mecA strain extracted/> -T-SA23srRNA and/> -T-mecA plasmid, transcribe RNA from the plasmid, purify to remove DNA, and quantify and identify RNA.

Example 3 Real-time fluorescent nucleic acid isothermal amplification detection method for methicillin-resistant Staphylococcus aureus

1. Sample collection, transportation and storage

Sample collection

1.1 Collection of nasal and oropharyngeal swab specimens: Use a medical cotton swab to collect nasal and oropharyngeal secretions. Soak the swab head in 2 mL of normal saline and squeeze it dry against the tube wall. Add 2 mL of sample preservation solution and set aside. This sample is the sample to be tested.

1.2 Collection of wound secretion samples: Use a medical cotton swab to collect wound secretions, soak the swab head in 2mL of normal saline and squeeze it dry against the tube wall, add 2mL of sample preservation solution and set aside. This sample is the sample to be tested.

Specimen transportation and storage

The collected specimens can be used for testing immediately or stored at -70℃ for a long time. The storage period at -20℃ is 6 months. The specimens should avoid repeated freezing and thawing. The specimens are transported in 0℃ ice bottles.

2. Nucleic acid extraction

2.1 Add 400 μl of sample preservation solution (HEPES 50 mM, (NH ₄ ) ₂ SO ₄ 35 mM) and 400 μl of the sample to be tested (if insufficient, use saline to make up), into a sample processing tube (1.5 mL centrifuge tube), and use the sample preservation solution to lyse the methicillin-resistant Staphylococcus aureus in the sample to be tested to obtain a lysate containing methicillin-resistant Staphylococcus aureus nucleic acid.

2.2 Add 200 μl of nucleic acid extraction solution (HEPES 200 mM, EDTA 100 mM, LiCl 800 mM, capture probe 15 μM, magnetic beads 150 mg/L) and 20 μl of internal standard solution (containing 10 ⁸ copies/mL SA 23srRNA IC RNA and 10 ⁵ copies/mL mecA IC RNA RNA) to the sample processing tube (1.5 mL centrifuge tube), mix well, keep warm at 60°C for 5 minutes, leave at room temperature for 10 minutes, and divide the prepared liquid into two equal parts for use.

2.3 Place the sample processing tube on the magnetic bead separation device and let it stand for 5-10 minutes. After the magnetic beads are adsorbed on the tube wall, keep the sample processing tube on the magnetic bead separation device, discard the liquid, and retain the magnetic beads. Add 1mL of washing solution (HEPES 25mM, NaCl150mM, 1% SDS, EDTA 2.5mM;) shake evenly and let it stand for 5-10 minutes, discard the liquid, retain the magnetic beads, and repeat 2 times.

2.4 Move the sample processing tube away from the magnetic bead separation device. The tube contains the magnetic bead-nucleic acid complex for use (the magnetic beads should be clearly visible in this step).

In the above detection operation, the sample to be tested in step 2.1 is a culture of methicillin-resistant Staphylococcus aureus.

3. SAT nucleic acid amplification test

3.1 Add 40 μl amplification detection solution (40 μl reaction solution + 2.5 μl detection solution) to the sample processing tubes to wash the magnetic beads. The reaction solution specifically contains Tris 15 mM, MgCl ₂ 15 mM, dNTP 2.5 mM, NTP 3 mM, PVP40 1%, KCl 10 mM; the concentration of T7 primers in SA detection solution is 7.5 pmol/reaction, the concentration of nT7 primers is 7.5 pmol/reaction, the concentration of SA detection probes is 5 pmol/reaction, and the concentration of SA internal standard detection probes is 5 pmol/reaction; the concentration of T7 primers in mecA detection solution is 2.5 pmol/reaction, the concentration of nT7 primers is 5 pmol/reaction, the concentration of mecA detection probes is 5 pmol/reaction, and the concentration of mecA internal standard detection probes is 5 pmol/reaction.

3.2 Take 30 μl of the above reaction detection solution that has been shaken and mixed, add it to a clean micro-reaction tube, and use a 7500 PCR instrument (product of ABI, USA) to keep it at 60°C for 10 minutes and at 42°C for 5 minutes; add 10 μl of enzyme solution that has been preheated to 42°C to the micro-reaction tube, shake it at 1200 rpm for 15 seconds to mix it. The enzyme solution contains 1500 U/reaction of M-MLV reverse transcriptase, 1000 U/reaction of T7 RNA polymerase, 10 mM HEPES pH 7.5, 15 mM N-acetyl-L-cysteine, 0.15 mM zinc acetate, 20 mM trehalose, 100 mM Tris-HCl pH 8.0, 80 mM KCl, 0.25 mM EDTA, 0.5% (v/v) Triton X-100 and 30% (v/v) glycerol.

3.3 Quickly transfer the micro reaction tube to a constant temperature fluorescence detection instrument (ABI7500 fluorescence quantifier, ABI product), react at 42°C for 40 minutes, set the fluorescence to be detected once every 1 minute, and detect 40 times in total; select the FAM channel (target signal detection, F1) and the VIC channel (HEX and VIC have similar wavelengths, internal standard signal detection, F2) as the fluorescein channel.

4. Result determination

According to the curve obtained from the SAT amplification results, the threshold line is set, the dt value is read, and the result is determined.

Threshold setting: The threshold line just exceeds the highest point of the normal negative control amplification curve. dt represents the horizontal coordinate reading of the intersection of the sample curve and the threshold line (similar to the ct value of the general real-time fluorescence PCR experiment results)

①Result determination:

SA: F1 channel: samples with dt≤35 are SA positive; samples with 35＜dt＜40 are recommended to be retested, and the test results of F1 channel: samples with dt＜40 are SA positive.

mecA: F1 channel: samples with dt≤35 are mecA positive; samples with 35＜dt＜40 are recommended to be retested, and the test results of F1 channel: samples with dt＜40 are mecA positive.

② Result determination: If the dt value of F1 channel is not available or is 40, and the dt of F2 channel is ≤35, it is negative.

③Result determination:

Table 1

SA test results mecA test results MRSA result judgment + + + — + — + — — — — —

Quality control: The dt values of the positive control, critical weak positive control, and negative control should meet the following conditions at the same time; otherwise, the experiment will be considered invalid and must be repeated.

(1) SA positive control, SA critical weak positive control: F1 channel: positive control dt≤critical weak positive control dt≤35.

(2) SA negative control: F1 channel: dt has no value or is 40, while F2 channel: dt≤35.

(3) mecA positive control, mecA critical weak positive control: F1 channel: positive control dt≤critical weak positive control dt≤35.

(4) mecA negative control: F1 channel: dt has no value or is 40, while F2 channel: dt≤35.

This kit is designed for methicillin-resistant Staphylococcus aureus. It uses real-time fluorescence constant temperature amplification to detect RNA. It is simpler than conventional PCR operation, with higher sensitivity and specificity. It takes only 40 minutes to complete the amplification detection. Because the amplified product is RNA rather than DNA, it is easily degraded in the environment and has low pollution. Adding a competitive internal standard to the kit can further reduce false negative results caused by system inhibition, ensuring more accurate test results.

5. Results

The methicillin-resistant Staphylococcus aureus samples were numbered as methicillin-resistant Staphylococcus aureus culture or other bacterial culture specimens 1-7, and a negative control (a solution without the methicillin-resistant Staphylococcus aureus target nucleic acid (SA23srRNA and mecA RNA) sequence or without methicillin-resistant Staphylococcus aureus) and a positive control (a mixture of 10 ⁴ -10 ⁶ copies/mL SA 23s rRNA and 10 ⁴ -10 ⁶ copies/mL mecA gene in vitro transcribed RNA dilution) were set. The results of SA are shown in Figure 5 (F1 fluorescence channel) and Figure 6 (F2 fluorescence channel); mecA is shown in Figure 7 (F1 fluorescence channel) and Figure 8 (F2 fluorescence channel). According to the dt values of the F1 channel and the F2 channel, combined with the result judgment criteria, samples 1, 3, 4, 6, and 7 were judged to be positive, and samples 2 and 5 were negative. The results show that the kit of the present invention has high accuracy in detecting methicillin-resistant Staphylococcus aureus, and the on-machine amplification detection time only takes 40 minutes, and has the characteristics of short cycle, high sensitivity, high specificity, low pollution and stable reaction.

According to the disclosure of the present invention, a person skilled in the art can implement the universal real-time fluorescent nucleic acid constant temperature amplification detection kit for methicillin-resistant Staphylococcus aureus (MRSA) claimed in the present invention without too much experimentation, and achieve the expected effect. The embodiments disclosed in the present invention are only a detailed description of the present invention, but are not sufficient to limit the present invention. A person skilled in the art can replace the preparation described herein with obvious similar substitutes or modifications, or replace the preparations described herein with certain chemically or biologically structurally related preparations, or make changes to the relevant contents of the present invention, but they do not exceed the spirit, scope and thought of the present invention, and they all fall within the scope of protection claimed by the present invention.

Sequence Listing

<110> Shanghai Rendu Biotechnology Co., Ltd.

<120> A method for isothermal amplification of methicillin-resistant Staphylococcus aureus (MRSA) nucleic acid

<130> P2018-0782

<160> 23

<170> PatentIn version 3.5

<210> 1

<211> 116

<212> DNA/RNA

<213> Artificial sequence

<400> 1

agcgattgat ggtgatacgg ttaaattaat gtacaaaggt caaccaatga cattcagact 60

attattggtt gatacacctg aaacaaagca tcctaaaaaa ggtgtagaga aatatg 116

<210> 2

<211> 125

<212> DNA/RNA

<213> Artificial sequence

<400> 2

aatcagaacg tggtaaaatt ttagaccgaa acaatgtgga attggccaat acaggaacag 60

catatgagat aggcatcgtt ccaaagaatg tatctaaaaa agattataaa gcaatcgcta 120

aagaa 125

<210> 3

<211> 54

<212> DNA/RNA

<213> Artificial sequence

<400> 3

tgcacaccgg atggccaatc caataaaaaa aaaaaaaaaa aaaaaaaaa aaaa 54

<210> 4

<211> 46

<212> DNA/RNA

<213> Artificial sequence

<400> 4

aatttaatac gactcactat agggagacat atttctctac accttt 46

<210> 5

<211> 22

<212> DNA/RNA

<213> Artificial sequence

<400> 5

agcgattgat ggtgatacgg tt 22

<210> 6

<211> 51

<212> DNA/RNA

<213> Artificial sequence

<400> 6

aatttaatac gactcactat agggagattc tttagcgatt gctttataat c 51

<210> 7

<211> 18

<212> DNA/RNA

<213> Artificial sequence

<400> 7

aatcagaacg tggtaaaa 18

<210> 8

<211> 26

<212> DNA/RNA

<213> Artificial sequence

<400> 8

ccgucauuca gacuauuauu ggacgg 26

<210> 9

<211> 27

<212> DNA/RNA

<213> Artificial sequence

<400> 9

cacaucagga acagcauaug agaugug 27

<210> 10

<211> 434

<212> DNA/RNA

<213> Artificial sequence

<400> 10

cgtaaataga agtggttctg aagatccaac agtatatagt gcaacttcaa ctaaaaaatt 60

acataaagaa cctgcgacat taattaaagc gattgatggt gatacggtta aattaatgta 120

caaaggtcaa ccaatgacat tcagactatt attggttgat acacctgaaa caaagcatcc 180

taaaaaaggt gtagagaaat atggtcctga agcaagtgca tttacgaaaa aaatggtaga 240

aaatgcaaag aaaattgaag tcgagtttga caaaggtcaa agaactgata aatatggacg 300

tggcttagcg tatatttatg ctgatggaaa aatggtaaac gaagctttag ttcgtcaagg 360

cttggctaaa gttgcttatg tttataaacc taacaataca catgaacaac ttttaagaaa 420

aagtgaagca caag 434

<210> 11

<211> 424

<212> DNA/RNA

<213> Artificial sequence

<400> 11

aggcgttaaa gatataaaca ttcaggatcg taaaataaaa aaagtatcta aaaataaaaa 60

acgagtagat gctcaatata aaattaaaac aaactacggt aacattgatc gcaacgttca 120

atttaatttt gttaaagaag atggtatgtg gaagttagat tgggatcata gcgtcattat 180

tccaggaatg cagaaagacc aaagcataca tattgaaaat ttaaaatcag aacgtggtaa 240

aattttagac cgaaacaatg tggaattggc caatacagga acagcatatg agataggcat 300

cgttccaaag aatgtatcta aaaaagatta taaagcaatc gctaaagaac taagtatttc 360

tgaagactat atcaaacaac aaatggatca aaattgggta caagatgata ccttcgttcc 420

actt 424

<210> 12

<211> 430

<212> DNA/RNA

<213> Artificial sequence

<400> 12

cgtaaataga agtggttctg aagatccaac agtatatagt gcaacttcaa ctaaaaaatt 60

acataaagaa cctgcgacat taattaaagc gattgatggt gatacggtta aattaatgta 120

caaaggtcaa ccaatgacag ugauacgaga gttgatacac ctgaaacaaa gcatcctaaa 180

aaaggtgtag agaaatatgg tcctgaagca agtgcattta cgaaaaaaat ggtagaaaat 240

gcaaagaaaa ttgaagtcga gtttgacaaa ggtcaaagaa ctgataaata tggacgtggc 300

ttagcgtata tttatgctga tggaaaaatg gtaaacgaag ctttagttcg tcaaggcttg 360

gctaaagttg cttatgttta taaacctaac aatacacatg aacaactttt aagaaaaagt 420

gaagcacaag 430

<210> 13

<211> 419

<212> DNA/RNA

<213> Artificial sequence

<400> 13

aggcgttaaa gatataaaca ttcaggatcg taaaataaaa aaagtatcta aaaataaaaa 60

acgagtagat gctcaatata aaattaaaac aaactacggt aacattgatc gcaacgttca 120

atttaatttt gttaaagaag atggtatgtg gaagttagat tgggatcata gcgtcattat 180

tccaggaatg cagaaagacc aaagcataca tattgaaaat ttaaaatcag aacgtggtaa 240

aattttagac cgaaacaatg tggaattggc caataccagg uaucaguata ggcatcgttc 300

caaagaatgt atctaaaaaa gattataaag caatcgctaa agaactaagt atttctgaag 360

actatatcaa acaacaaatg gatcaaaatt gggtacaaga tgataccttc gttccactt 419

<210> 14

<211> 22

<212> DNA/RNA

<213> Artificial sequence

<400> 14

ccgacaguga uacgagagtc gg 22

<210> 15

<211> 22

<212> DNA/RNA

<213> Artificial sequence

<400> 15

ccgacccagg uaucagugtc gg 22

<210> 16

<211> 112

<212> DNA/RNA

<213> Artificial sequence

<400> 16

agcgattgat ggtgatacgg ttaaattaat gtacaaaggt caaccaatga cagugauacg 60

agagttgata cacctgaaac aaagcatcct aaaaaaggtg tagagaaata tg 112

<210> 17

<211> 120

<212> DNA/RNA

<213> Artificial sequence

<400> 17

aatcagaacg tggtaaaatt ttagaccgaa acaatgtgga attggccaat accagguauc 60

aguataggca tcgttccaaa gaatgtatct aaaaaagatt ataaagcaat cgctaaagaa 120

<210> 18

<211> 48

<212> DNA/RNA

<213> Artificial sequence

<400> 18

aatttaatac gactcactat agggagaccg tatcaccatc aatcgctt 48

<210> 19

<211> 19

<212> DNA/RNA

<213> Artificial sequence

<400> 19

caaatgcatc acaaacaga 19

<210> 20

<211> 25

<212> DNA/RNA

<213> Artificial sequence

<400> 20

cgcucugaag auccaacagu gagcg 25

<210> 21

<211> 44

<212> DNA/RNA

<213> Artificial sequence

<400> 21

aatttaatac gactcactat agggagaatg ctttggtctt tctg 44

<210> 22

<211> 20

<212> DNA/RNA

<213> Artificial sequence

<400> 22

ctacggtaac attgatcgca 20

<210> 23

<211> 27

<212> DNA/RNA

<213> Artificial sequence

<400> 23

cgccggugga aguuagauug ggcggcg 27

Claims (3)

1. A combination of primers and probes, characterized in that the primers are specific primer pairs for mecA RNA of methicillin-resistant Staphylococcus aureus MRSA, and the primer pairs are: T7 primer for mecA RNA: the sequence is shown in SEQ ID NO: 6; and nT7 primer for mecA RNA: the sequence is shown in SEQ ID NO: 7; And the sequence of the probe is shown in SEQ ID NO:9. 2. Use of the combination of primers and probes according to claim 1, characterized in that it is used to prepare a detection kit or a detection reagent for detecting methicillin-resistant Staphylococcus aureus (MRSA). 3. The use according to claim 2, characterized in that the detection kit or detection reagent is used to detect whether methicillin-resistant Staphylococcus aureus (MRSA) exists in human nasal and pharyngeal swabs and wound secretions.