CN114717339B - Application of reagent for detecting SNP locus in preparation of kit - Google Patents
Application of reagent for detecting SNP locus in preparation of kit Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 7
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- WKDDRNSBRWANNC-UHFFFAOYSA-N Thienamycin Natural products C1C(SCCN)=C(C(O)=O)N2C(=O)C(C(O)C)C21 WKDDRNSBRWANNC-UHFFFAOYSA-N 0.000 description 4
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention provides a use of a reagent for detecting SNP loci in preparation of a kit for determining a drug sensitive phenotype of klebsiella pneumoniae, wherein the SNP loci comprise at least one of the following: the klebsiella pneumoniae genome HS11286 is taken as a reference genome, and the 218514 th, 4907417 th and 5141114 th are taken as reference genome.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to application of a reagent for detecting SNP loci in preparation of a kit, and more particularly relates to a method for determining a Klebsiella pneumoniae drug-sensitive phenotype, a kit, an analysis device for determining the Klebsiella pneumoniae drug-sensitive phenotype and equipment for guiding drug use.
Background
Klebsiella pneumoniae is a common hospital-related pathogen, considered to be an urgent threat to human health, and is one of the most alarming pathogens in antibiotic resistance, which is associated with some very important multi-drug resistant (MDR) pathogens and is called ESKAPE (enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter).
With the widespread use of antibiotics, the clinical multi-drug resistance rate of klebsiella pneumoniae is gradually increased. The drug resistance rate of the drug resistance agent for the final line of carbapenems for treating super drug-resistant bacteria is increased year by year, which leads to the situation that no drug is available for clinical treatment; meanwhile, the basic method for clinically identifying drug resistance at present mainly comprises the steps of determining the minimum inhibitory concentration of bacteria based on culture, and has the problems of low culture positive rate and long time consumption. In addition, the existing PCR detection-based kits on the market mainly detect certain enzyme genes specific to bacteria, and have the limitation that the kit cannot cover all drug-resistant genes.
Therefore, it is important to develop a method that can achieve rapid and comprehensive detection of the drug-sensitive phenotype of klebsiella pneumoniae.
Disclosure of Invention
In a first aspect of the invention, the invention proposes the use of a reagent for detecting SNP sites in the preparation of a kit for determining the drug sensitive phenotype of Klebsiella pneumoniae. According to an embodiment of the invention, the SNP site comprises at least one of the following: the klebsiella pneumoniae genome HS11286 is taken as a reference genome, and the 218514 th, 4907417 th and 5141114 th are taken as reference genome. According to the embodiment of the invention, the base of the SNP locus in the genome of the strain to be detected is detected, so that whether the strain to be detected belongs to a drug-resistant strain can be determined efficiently and accurately.
In a second aspect of the invention, the invention provides a method of determining a drug-sensitive phenotype of klebsiella pneumoniae. According to an embodiment of the invention, the method comprises: (1) Determining a base type of a predetermined SNP site, wherein the predetermined SNP site comprises at least one selected from the group consisting of: taking klebsiella pneumoniae genome HS11286 as a reference genome, wherein 218514 th, 4907417 th and 5141114 th are adopted; (2) And determining the drug sensitive phenotype of the strain to be tested based on the base type. According to the method provided by the embodiment of the invention, the genome of the strain to be detected is analyzed, whether the strain to be detected belongs to a drug-resistant strain can be determined through the specific base type of the SNP locus, and other specific SNP loci can be further analyzed, so that the drug resistance classification of the strain to be detected can be further determined, and the drug resistance information of each drug is obtained.
In a third aspect of the invention, the invention proposes a kit for determining a drug-sensitive phenotype of klebsiella pneumoniae, characterized by comprising primers and/or probes, wherein the probes and/or primers are used for identifying and/or amplifying at least one of the following SNP sites: the klebsiella pneumoniae genome HS11286 is taken as a reference genome, and the 218514 th, 4907417 th and 5141114 th are taken as reference genome. The kit provided by the embodiment of the invention can detect the sites, so that the evolutionary branches of the strain to be detected can be efficiently and accurately determined, and whether the strain belongs to the drug-resistant strain or not can be judged.
In a fourth aspect of the present invention, the present invention provides an assay device for determining a drug-sensitive phenotype of klebsiella pneumoniae. According to an embodiment of the invention, the apparatus comprises: a SNP site determining unit that determines a base type of a predetermined SNP site, wherein the predetermined SNP site includes at least one selected from the group consisting of: taking klebsiella pneumoniae genome HS11286 as a reference genome, wherein 218514 th, 4907417 th and 5141114 th are adopted; and a drug sensitive phenotype determining unit that determines a drug sensitive phenotype of the strain to be tested based on the base type. The analysis device according to the embodiment of the invention utilizes the method provided in the second aspect of the invention to identify the drug sensitive phenotype of the strain to be tested and judge whether the strain to be tested belongs to the drug resistant strain.
In a fifth aspect of the invention, the invention provides a device for guiding administration of a medicament for determining a medicament for treating a klebsiella pneumoniae-related disorder. According to an embodiment of the invention, the apparatus comprises: an analysis device according to a fourth aspect of the present invention; and a drug determination device that determines a drug for treating klebsiella pneumoniae based on a result of the analysis device. According to the device for guiding drug administration, disclosed by the embodiment of the invention, the drug resistance information of the klebsiella pneumoniae infected by a patient can be determined, the drug is selected according to the drug resistance rate of the klebsiella pneumoniae to the drug, the drug with high sensitivity can be selected in a targeted manner, the klebsiella pneumoniae can be quickly and effectively inhibited, the purpose of quick and effective treatment is achieved, and meanwhile, the treatment delay caused by the use of the drug with low sensitivity can be avoided.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a diagram showing the system evolution relationship and drug resistance spectrum information of KP1 according to an embodiment of the present invention, wherein the drug resistance spectrum represents AK/ATM/CFP/CIP/CRO/ETP/GEN/IPM/LVX/SXT/TOB from inside to outside, wherein each sub-leaf node represents a sample, white represents sensitivity, and black represents drug resistance;
FIG. 2 is a diagram showing the system evolution relationship and drug resistance spectrum information of Group1 according to an embodiment of the present invention, wherein the drug resistance spectrum represents AK/ATM/CFP/CIP/CRO/ETP/GEN/IPM/LVX/SXT/TOB from inside to outside, wherein each sub-leaf node represents a sample, white represents sensitivity, and black represents drug resistance;
FIG. 3 is a schematic diagram of an analytical device for determining a drug-sensitive phenotype of Klebsiella pneumoniae in accordance with an embodiment of the invention;
fig. 4 is a schematic diagram of an apparatus for directing medication according to an embodiment of the present invention.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
It should be noted that "SNP site" as used in the present invention means a single nucleotide polymorphism site differing from Klebsiella pneumoniae genome HS 11286. In a first aspect, the invention proposes the use of a reagent for detecting a SNP site in the preparation of a kit for determining the drug sensitive phenotype of klebsiella pneumoniae, characterized in that the SNP site comprises at least one of the following: the klebsiella pneumoniae genome HS11286 is taken as a reference genome, and the 218514 th, 4907417 th and 5141114 th are taken as reference genome. The inventor utilizes the existing strain genome of 2676 Klebsiella pneumoniae to construct a drug-resistant system evolution relation, finds out the SNP locus according to a drug-sensitive phenotype experiment of a drug, and determines specific base types of a strain cluster (Group 1) with concentrated drug-resistant information distribution and a strain cluster (Group 0) with scattered drug-resistant information distribution, so that whether the strain to be tested belongs to the drug-resistant strain can be determined efficiently and accurately according to the base information of the SNP locus of the strain to be tested. The two Group specific combinations of sites are shown in Table 1 in the order 218514, 4907417, 5141114.
TABLE 1
According to an embodiment of the present invention, the SNP site further includes at least one selected from the SNP sites listed in table 2. For example, when the base at the 49614 site is T, 100% is clone 1; when the base of the locus is C, 100% is clone 2; when the base at this site is A, 100% is clone 3. According to the base condition of the locus, one of three clips belonging to different drug-resistant clusters can be judged, and the rest 25 loci are sequentially analogized. Depending on the base of any of the 26 sites, the drug resistant clusters can be distinguished by 100%.
TABLE 2
According to the examples of the present invention, the inventors found a combination of 3 sites for the systematic evolution relationship and drug sensitive phenotype results of 2676 klebsiella pneumoniae to distinguish large clusters with drug resistant relationship (table 1). The large clusters are divided into Group1 with high drug resistance and concentrated distribution of drug resistance samples and Group0 with low drug resistance and dispersed distribution of drug resistance samples.
According to the embodiment of the invention, the inventor further divides the samples in the Group1 into 3 branches of the Group1, the Group 2 and the Group 3 with drug resistance according to the system evolution relation and the drug sensitivity detection result. Based on this, some SNP molecular markers capable of specifically distinguishing three clades were found from the sample genome (Table 2).
According to a specific embodiment of the invention, a system evolution relationship is constructed: the invention is based on a sample of the evolutionary branch KP1 with strongest drug resistance in 2676 Klebsiella pneumoniae collected from 2013 to 2018 in a drug-resistant bacteria library of a certain hospital in China. Firstly, carrying out whole genome sequencing on all samples, carrying out SNP (single nucleotide polymorphism) rolling, and constructing a system evolution relation according to the obtained SNP matrix. Drug resistance and sensitive phenotype acquisition of the strain: drug resistance and susceptibility phenotypes of strains were identified for 11 of the following drugs including Amikacin (AK), aztreonam (ATM), cefoperazone (CFP), ciprofloxacin (CIP), ceftriaxone (CRO), ertapenem (ETP), gentamicin (GEN), imipenem (IPM), levofloxacin (LVX), sulfamethoxazole-trimethoprim (SXT), tobramycin (TOB) for all samples. And screening large clusters with obvious drug resistance characteristics by combining the systematic evolution relation and drug resistance information of the sample, and finding a combination consisting of 3 sites to distinguish the large clusters with the drug resistance relation (table 1). The large clusters are divided into Group1 with concentrated distribution of drug resistance information and Group0 with dispersed distribution of drug resistance rate information (figure 1). According to the guidelines of the american society for Infectious Diseases (IDSA), if a strain has a drug resistance of more than 20% to a drug, the drug is not recommended for treating the disease caused by the strain. Based on the principle, the samples in Group1 can be divided into 3 branches of clone 1, clone 2 and clone 3 with drug resistance according to the system evolution relation and drug sensitivity detection result. Based on this, some SNP molecular markers capable of specifically distinguishing three clades were found from the sample genome. The screening method is as follows: based on SNP table obtained by SNP sizing, specific sites capable of strictly distinguishing three clades are screened out through python script. For example, 100% of this site is a base in clone 1, 100% is another base in clone 2, and 100% is another base in clone 3. The inventor selects 26 important SNP loci altogether, and forms our SNP molecular markers for distinguishing drug-resistant clusters (figure 2). Identification of 3 branch-specific SNP markers: the genome-wide SNP sites of all samples in Group1 were traversed and the marker sites of each clone that were specifically distinguishable from the other two clones were screened. That is, 100% of samples in clone 1 are a base, 100% are another base in clone 2, and 100% are another base in clone 3 at this site.
In a second aspect, the invention provides a method of determining a drug-sensitive phenotype of klebsiella pneumoniae comprising: (1) Determining a base type of a predetermined SNP site, wherein the predetermined SNP site comprises at least one selected from the group consisting of: taking klebsiella pneumoniae genome HS11286 as a reference genome, wherein 218514 th, 4907417 th and 5141114 th are adopted; and (2) determining the drug sensitive phenotype of the strain to be tested based on the base type. According to the method provided by the embodiment of the invention, whether the strain of the Klebsiella pneumoniae to be detected belongs to a drug-resistant strain can be rapidly and effectively identified, and further analysis and research can be carried out on the strain or a more accurate medication scheme can be made.
According to the embodiment of the invention, the method can be used for screening medicines, for example, the method can be used for determining the medicine resistance of the strain and the medicine resistance rate of the strain to certain type of antibiotics, and whether a new medicine to be detected is effective to a specific strain of Klebsiella pneumoniae or not is predicted based on the medicine resistance mechanism of microorganisms to the specific type of antibiotics.
According to the embodiment of the invention, when the bases at the 218514, 4907417 and 5141114 sites are T A T in sequence, the indication of drug resistance of the strain to be tested is provided.
According to the embodiment of the invention, when the bases at the 218514, 4907417 and 5141114 sites are C, C and C in sequence, the indication that the strain to be tested has no drug resistance is provided.
According to the embodiment of the invention, when the bases at the 218514, 4907417 and 5141114 sites are T C C in turn, the indication that the strain to be tested has no drug resistance is provided.
According to the embodiment of the invention, when the bases at the 218514, 4907417 and 5141114 sites are C, C and T in sequence, the indication that the strain to be tested has no drug resistance is provided.
According to the embodiment of the invention, when the bases at the 218514, 4907417 and 5141114 sites are C AC in sequence, the indication that the strain to be tested is not resistant is provided.
According to the embodiment of the invention, when the bases of the 218514, 4907417 and 5141114 sites are T C T in turn, the indication that the strain to be tested is not drug resistant is provided.
According to the embodiment of the invention, when the bases AT the 218514, 4907417 and 5141114 sites are C AT in sequence, the indication that the strain to be tested is not resistant is provided.
According to the embodiment of the invention, when the bases at the 218514, 4907417 and 5141114 sites are T AC in sequence, the indication that the strain to be tested is not drug resistant is provided.
According to an embodiment of the present invention, the strain to be tested is classified as Group1 when it is judged to be resistant according to the above criteria, and classified as Group0 when it is judged to be not resistant according to the above criteria.
According to the embodiment of the invention, the Group0 drug resistance rate is relatively low, and drug resistance samples are distributed in a system evolution relationship, so that when the strain to be detected is judged to be Group0, a traditional drug sensitivity experiment can be performed to determine the drug resistance of the strain to be detected.
According to the embodiment of the present invention, when the strain to be tested is judged as Group1, its evolutionary branches (clone 1, clone 2, clone 3) can be further determined according to the base types of the SNP sites shown in Table 2. According to an embodiment of the invention, when the base of the 49614 locus is T, the strain sample is classified as class 1; when the base at this site is C, the strain sample is classified as clone 2; when the base at this site is A, the strain sample is classified as clone 3.
According to the embodiment of the invention, any site or any site combination in the table 2 can be used for judging the attribution of the evolution branch of the sample to be detected, and judging whether the sample is the clone 1, the clone 2 or the clone 3, the inventor finds out that the 4839205 site is the most accurate and sensitive through a large number of researches, and can accurately distinguish the evolution branch of the sample to be detected, thereby judging the drug resistance property of the sample. When the base of the 4839205 locus is T, classifying the strain sample into a clone 1; when the base at this site is C, the strain sample is classified as clone 2; when the base at this site is A, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 155494 locus is A, the strain sample is classified as clone 1; when the base at this site is C, the strain sample is classified as clone 2; when the base at this site is T, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 283083 locus is T, the strain sample is classified as class 1; when the base at this site is G, the strain sample is classified as clone 2; when the base at this site is C, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 449376 locus is T, the strain sample is classified as class 1; when the base at this site is C, the strain sample is classified as clone 2; when the base at this site is G, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 495369 locus is a, the strain sample is classified as clone 1; when the base at this site is C, the strain sample is classified as clone 2; when the base at this site is T, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 573182 locus is T, the strain sample is classified as class 1; when the base at this site is C, the strain sample is classified as clone 2; when the base at this site is A, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 573299 locus is G, the strain sample is classified as clone 1; when the base at this site is C, the strain sample is classified as clone 2; when the base at this site is T, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 577551 locus is a, the strain sample is classified as clone 1; when the base at this site is C, the strain sample is classified as clone 2; when the base at this site is T, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 775891 locus is C, the strain sample is classified as clone 1; when the base at this site is G, the strain sample is classified as clone 2; when the base at this site is A, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 1075460 locus is a, the strain sample is classified as clone 1; when the base at this site is G, the strain sample is classified as clone 2; when the base at this site is T, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 1116497 locus is T, the strain sample is classified as class 1; when the base at this site is C, the strain sample is classified as clone 2; when the base at this site is A, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 1177312 locus is T, the strain sample is classified as class 1; when the base at this site is C, the strain sample is classified as clone 2; when the base at this site is G, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 1222291 locus is C, the strain sample is classified as clone 1; when the base at the site is T, the strain sample is classified into class 2; when the base at this site is G, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 1284861 locus is T, the strain sample is classified as class 1; when the base at the site is A, the strain sample is classified into class 2; when the base at this site is G, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 1580566 locus is a, the strain sample is classified as clone 1; when the base at this site is G, the strain sample is classified as clone 2; when the base at this site is T, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 1662442 locus is C, the strain sample is classified as clone 1; when the base at the site is A, the strain sample is classified into class 2; when the base at this site is T, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 2055657 locus is C, the strain sample is classified as clone 1; when the base at the site is T, the strain sample is classified into class 2; when the base at this site is G, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 3989835 locus is G, the strain sample is classified as clone 1; when the base at this site is C, the strain sample is classified as clone 2; when the base at this site is A, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 4088637 locus is G, the strain sample is classified as clone 1; when the base at this site is C, the strain sample is classified as clone 2; when the base at this site is A, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 4150905 locus is G, the strain sample is classified as clone 1; when the base at this site is C, the strain sample is classified as clone 2; when the base at this site is T, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 4494702 locus is G, the strain sample is classified as clone 1; when the base at the site is A, the strain sample is classified into class 2; when the base at this site is C, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 5101801 locus is G, the strain sample is classified as clone 1; when the base at the site is T, the strain sample is classified into class 2; when the base at this site is A, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 5123449 locus is C, the strain sample is classified as clone 1; when the base at the site is T, the strain sample is classified into class 2; when the base at this site is G, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 5128843 locus is C, the strain sample is classified as clone 1; when the base at this site is G, the strain sample is classified as clone 2; when the base at this site is A, the strain sample is classified as clone 3.
According to an embodiment of the invention, when the base of the 5154735 locus is a, the strain sample is classified as clone 1; when the base at the site is T, the strain sample is classified into class 2; when the base at this site is C, the strain sample is classified as clone 3.
It should be noted that, when constructing classification, the inventors divide the samples in Group1 into 3 branches of clone 1, clone 2 and clone 3 with drug resistance according to the system evolution relationship and drug sensitivity detection result, and the SNP loci shown in Table 2 and the bases corresponding to the SNP loci are SNP molecular markers for distinguishing the specificity of the three branches.
According to an embodiment of the present invention, after step (2), the method further comprises determining the drug resistance rate of the strain to be tested based on table 2. The strain to be tested was judged for resistance to 11 drugs according to classification of clade, as shown in Table 3.
Table 3: drug resistance per coat for each drug
According to the embodiment of the invention, the strain classified as the clone 1 has higher drug resistance, the average drug resistance is about 85%, the strain classified as the clone 2 has lower drug resistance, the average drug resistance is about 5%, and the strain classified as the clone 3 has the next highest drug resistance, and the average drug resistance is about 60%. The drug resistance of each drug still needs to be judged one by one according to the table three. In a third aspect of the invention, the invention proposes a kit for determining a drug-sensitive phenotype of klebsiella pneumoniae, characterized by comprising primers and/or probes, wherein the probes and/or primers are used for identifying and/or amplifying at least one of the following SNP sites: the klebsiella pneumoniae genome HS11286 is taken as a reference genome, and the 218514 th, 4907417 th and 5141114 th are taken as reference genome. According to the embodiment of the invention, the SNP locus of the strain to be detected is obtained by using a kit and the like, so that branches of the strain to be detected can be distinguished, and the drug resistance condition of the strain to be detected can be judged by classification.
According to an embodiment of the present invention, the kit further comprises a probe that can recognize at least one of the SNP sites set forth in Table 2.
According to the embodiment of the invention, the probe can further comprise a fluorescent group, and the specific probe can emit fluorescence when combined with a specific SNP locus, so that the base type of the SNP locus of the strain to be detected is determined, and the drug resistance information of the strain to be detected is judged. The probe can also be used for target capture sequencing, specifically obtains the sequence information of the SNP locus of the strain to be detected, and further judges the drug resistance information of the strain to be detected.
According to an embodiment of the present invention, the kit further comprises primers capable of amplifying at least one of the SNP sites set forth in Table 2. The primer can specifically amplify the SNP locus, and sequence information of the SNP locus is obtained by utilizing a sequencing technology, so that drug resistance information of the strain to be detected is judged.
It should be noted that the "sequencing" or "sequencing technique" referred to in the present invention refers to a method for obtaining a nucleic acid sequence, and is not particularly limited, and may be sanger sequencing, high throughput sequencing, second generation sequencing, single molecule sequencing, or the like.
In a fourth aspect of the invention, the invention provides an assay device for determining a drug-sensitive phenotype of klebsiella pneumoniae. According to an embodiment of the invention, as illustrated in fig. 3, the analysis device comprises:
a SNP site determination unit 100 that determines a base type of a predetermined SNP site, wherein the predetermined SNP site includes at least one selected from the following: taking klebsiella pneumoniae genome HS11286 as a reference genome, wherein 218514 th, 4907417 th and 5141114 th are adopted; and a drug susceptibility phenotype determining unit 200, the drug susceptibility phenotype determining unit 200 being connected to the SNP locus determining unit 100 for determining the drug susceptibility phenotype of the strain to be tested based on the base type.
According to an embodiment of the present invention, as shown in fig. 3, the apparatus further includes: the drug resistance rate determination unit 300 makes a drug resistance rate judgment for the strain to be tested based on table 2.
According to an embodiment of the present invention, the analysis device is designed according to the method according to the second aspect of the present invention, and the biological principle is the same as that of the method according to the second aspect of the present invention, and will not be described herein.
In a fifth aspect of the invention, the invention provides a device for guiding administration of a medicament for determining a medicament for treating a klebsiella pneumoniae-related disorder. According to an embodiment of the invention, as shown in fig. 4, the apparatus comprises: the analysis device 1000 proposed in the fourth aspect of the present invention; and a drug determining device 2000 connected to the analyzing device 1000, the drug determining device determining a drug for treating klebsiella pneumoniae based on a result of the analyzing device.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not limiting in any way.
Examples
(1) After the Klebsiella pneumoniae sample to be detected is obtained, whole genome sequencing is performed on the Klebsiella pneumoniae sample, and SNP (single nucleotide polymorphism) cloning is performed by taking Klebsiella pneumoniae genome HS11286 (Genebank Accession:NC_ 016845) as a reference.
(2) According to the result of SNP rolling, group1 with high drug resistance and concentrated distribution of drug resistance samples and Group0 with low drug resistance and dispersed distribution of drug resistance samples can be distinguished based on 3 sites (218514, 4907417, 5141114) of the reference sequence HS11286, and the combination of two Group-specific sites is shown in Table 1 in the order of 218514, 4907417, 5141114. Group1 and Group0 can be distinguished based on the combination of sites in the bit sequences (218514, 4907417, 5141114) in table 1. For example: if the genome of the sample is T, A, T at positions 218514, 4907417, 5141114 in that order, the sample can be classified as Group1. Because Group0 resistance is relatively low and the drug resistant samples are scattered throughout the system evolution, it is recommended to conduct traditional drug susceptibility experiments on samples classified as Group0 to determine their resistance. Samples classified as Group1 may then continue to be classified according to SNP molecular markers.
(3) In the samples of Group1, group1 can be divided into 3 clips based on the following sites on the reference sequence HS 11286. As shown in table 2. For example: if a sample of Group1 has been determined to be "T" at position 49614 or to correspond to a specific SNP molecular marker for other clades 1 in Table 2, the sample can be classified as a clade1.Clade2 and Clade3 classification methods and so on.
(4) The drug resistance of the sample to 11 drugs can be judged according to classification of the class. The sample resistance is counted in table 3.
According to the guidelines of the American society for Infectious Diseases (IDSA), if a certain antibiotic has a drug resistance of more than 20% to a certain strain, the antibiotic cannot be used to treat a disease caused by the strain. Based on the statistical results of 1064 samples, the strain with the clone 1 had a higher drug resistance, the average drug resistance was about 85%, the strain classified as clone 2 had a lower drug resistance, the average drug resistance was about 5%, the strain classified as clone 3 had a next higher drug resistance, and the average drug resistance was about 60%.
If it is necessary to determine the drug resistance rate of a certain drug according to the application scenario, the analysis can be further performed based on table 3. For example: for AK (amikacin), the drug resistance of the clade2 and 3 is lower and is classified as sensitivity, and the drug resistance of the clade1 as the clade with the highest drug resistance degree shown by the drug resistance spectrum is lower than that of other drugs; for ATM (aztreonam), only the clone 2 is sensitive, the clone 3 is classified as drug resistance when the drug resistance threshold is exceeded, the clone 1 drug resistance rate is over 90%, and the drug resistance rate is extremely high; for SXT (sulfamethoxazole-trimethoprim), clone 2 still showed sensitivity, and clone 1 and clone 3 both showed some degree of resistance.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (8)
1. Use of a reagent for detecting a SNP site in the preparation of a kit for determining a drug sensitive phenotype of klebsiella pneumoniae, characterized in that the SNP site comprises the following: taking klebsiella pneumoniae genome HS11286 as a reference genome, 218514 th, 4907417 th and 5141114 th,
when the bases of the 218514, 4907417 and 5141114 sites are T A T in sequence, the indication of drug resistance of the strain to be tested is provided,
when the bases of the 218514, 4907417 and 5141114 loci are C, C and C in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 loci are T C C in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 loci are C C T in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 loci are C A C in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 sites are sequentially T C T, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 loci are C A T in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 sites are T A C in sequence, the indication that the strain to be tested has no drug resistance is provided.
2. The use according to claim 1, wherein the SNP site further comprises at least one selected from the SNP sites listed in table 2;
TABLE 2
。
3. A method for determining a drug-sensitive phenotype of klebsiella pneumoniae for non-disease diagnostic use, comprising:
(1) Determining the base type of a predetermined SNP site, wherein the predetermined SNP site comprises the following: taking klebsiella pneumoniae genome HS11286 as a reference genome, wherein 218514 th, 4907417 th and 5141114 th are positions; and
(2) Determining the drug sensitive phenotype of the strain to be tested based on the base type;
when the bases of the 218514, 4907417 and 5141114 loci are T A T in sequence, the indication of drug resistance of the strain to be detected is provided;
when the bases of the 218514, 4907417 and 5141114 loci are C, C and C in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 loci are T C C in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 loci are C C T in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 loci are C A C in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 sites are sequentially T C T, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 loci are C A T in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 sites are T A C in sequence, the indication that the strain to be tested has no drug resistance is provided.
4. The method of claim 3, wherein the SNP sites further comprise at least one selected from the SNP sites listed in table 2;
TABLE 2
。
5. The method of claim 3, further comprising, after step (2), making a drug resistance determination for the test strain based on table 2;
TABLE 2
。
6. An analytical device for determining a drug-sensitive phenotype of klebsiella pneumoniae, comprising:
a SNP site determination unit that determines a base type of a predetermined SNP site, wherein the predetermined SNP site comprises the following: taking klebsiella pneumoniae genome HS11286 as a reference genome, wherein 218514 th, 4907417 th and 5141114 th are positions;
a drug sensitive phenotype determining unit that determines a drug sensitive phenotype of the strain to be tested based on the base type;
when the bases of the 218514, 4907417 and 5141114 sites are T A T in sequence, the indication of drug resistance of the strain to be tested is provided,
when the bases of the 218514, 4907417 and 5141114 loci are C, C and C in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 loci are T C C in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 loci are C C T in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 loci are C A C in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 sites are sequentially T C T, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 loci are C A T in sequence, the indication that the strain to be detected does not have drug resistance is provided;
when the bases of the 218514, 4907417 and 5141114 sites are T A C in sequence, the indication that the strain to be tested has no drug resistance is provided.
7. The apparatus as recited in claim 6, further comprising:
a drug resistance rate determination unit that makes a drug resistance rate judgment for the strain to be tested based on table 2;
TABLE 2
。
8. An apparatus for directing the administration of a drug for determining a drug for treating a klebsiella pneumoniae-related disorder, comprising:
the analysis device of claim 6 or 7; and
and a drug determination device that determines a drug for treating klebsiella pneumoniae based on a result of the analysis device.
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