CN114717339A - Application of reagent for detecting SNP (Single nucleotide polymorphism) sites in preparation of kit - Google Patents
Application of reagent for detecting SNP (Single nucleotide polymorphism) sites in preparation of kit Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- 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
- 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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- C—CHEMISTRY; METALLURGY
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention provides application of a reagent for detecting SNP sites in preparation of a kit for determining drug sensitive phenotype of Klebsiella pneumoniae, wherein the SNP sites comprise at least one of the following: taking Klebsiella pneumoniae genome HS11286 as a reference genome, 218514 th, 4907417 th and 5141114 th.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to application of a reagent for detecting SNP (single nucleotide polymorphism) loci in preparation of a kit, and more particularly relates to a method for determining a drug sensitive phenotype of Klebsiella pneumoniae, the kit, an analysis device for determining the drug sensitive phenotype of the Klebsiella pneumoniae, and equipment for guiding medication.
Background
Klebsiella pneumoniae is a common hospital-associated pathogen, considered to be an imminent threat to human health, and one of the most alarming pathogens in antibiotic resistance, associated with several very important multidrug-resistant (MDR) pathogens and known as ESKAPE (enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter).
With the wide use of antibiotics, the clinical multidrug resistance rate of klebsiella pneumoniae is also gradually increasing. The drug resistance rate of the clinical commonly used ditridecylcephalosporin, aminoglycoside and fluoroquinolone drugs is higher, and the drug resistance rate of the last first-line carbapenem drug for treating super drug-resistant bacteria is also increased year by year, so that the situation that no drug is available for clinical treatment is caused; meanwhile, the current basic method for clinically identifying drug resistance mainly comprises the step 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-based detection kits on the market mainly aim at certain enzyme genes specific to bacteria for detection, and have the limitation that all drug-resistant genes cannot be covered.
Therefore, it is very important to develop a method for rapidly and comprehensively detecting 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 sites include at least one of: taking Klebsiella pneumoniae genome HS11286 as a reference genome, 218514 th, 4907417 th and 5141114 th. According to the embodiment of the invention, the base of the SNP locus in the genome of the strain to be detected is detected, and whether the strain to be detected belongs to the drug-resistant strain can be determined efficiently and accurately.
In a second aspect of the invention, the invention provides a method of determining the 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, 218514 th, 4907417 th and 5141114 th; (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 or not can be determined through the specific base type of the SNP locus, and other specific SNP loci can be further analyzed, the drug resistance classification of the strain to be detected is further determined, the information of the drug resistance rate of each drug is obtained, the method is efficient and convenient, specific genes do not need to be specified, the problem of multiple drug resistance classifications can be solved at one time, the drug resistance information is given, and clinical medication is guided.
In a third aspect, the present invention provides 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: taking Klebsiella pneumoniae genome HS11286 as a reference genome, 218514 th, 4907417 th and 5141114 th. The kit provided by the embodiment of the invention can detect the loci, so that the clade of the strain to be detected can be efficiently and accurately determined, and whether the strain belongs to a drug-resistant strain or not can be judged.
In a fourth aspect of the present invention, an assay device for determining the drug sensitive phenotype of klebsiella pneumoniae is presented. According to an embodiment of the invention, the apparatus comprises: an SNP site determination unit determining 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, 218514 th, 4907417 th and 5141114 th; and a drug sensitive phenotype determination 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 present invention identifies the drug sensitive phenotype of the strain to be tested by using the method set forth in the second aspect of the present invention, and determines whether the strain to be tested belongs to a drug-resistant strain.
In a fifth aspect of the invention, the invention proposes a device for guiding medication for determining a medication for the treatment of a klebsiella pneumoniae related disease. According to an embodiment of the invention, the apparatus comprises: the analysis device set forth in the 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 equipment for guiding medication, disclosed by the embodiment of the invention, the drug resistance information of the Klebsiella pneumoniae infected by a patient can be determined, and the drug selection is carried out according to the drug resistance rate of the Klebsiella pneumoniae, so that the drug with high sensitivity can be selected in a targeted manner, the Klebsiella pneumoniae can be inhibited quickly and effectively, the aim of quickly and effectively treating is fulfilled, 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 above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a diagram illustrating the system evolution and the 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, respectively, wherein each sub-leaf node represents a sample, white represents sensitivity, and black represents drug resistance;
FIG. 2 is the system evolution 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, each sub-leaf node represents a sample, white represents sensitivity, and black represents drug resistance;
FIG. 3 is a schematic view of an analysis apparatus for determining the drug sensitive phenotype of Klebsiella pneumoniae, in accordance with an embodiment of the present invention;
FIG. 4 is a schematic view of an apparatus for guiding administration of a medication according to an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are illustrative and intended to explain the present invention and should not be construed as limiting the present invention.
It should be noted that the "SNP site" used in the present invention refers to a single nucleotide polymorphism site that is different from the Klebsiella pneumoniae genome HS 11286. In a first aspect of the invention, the invention proposes the use of a reagent for detecting a SNP site for 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: taking Klebsiella pneumoniae genome HS11286 as a reference genome, 218514 th, 4907417 th and 5141114 th. The inventor utilizes the existing strain genome of 2676 Klebsiella pneumoniae to construct an evolutionary relationship of a drug resistance system, finds the SNP locus according to a drug sensitive phenotype experiment of a drug, and determines specific base types of a strain cluster (Group1) with concentrated drug resistance information and a strain cluster (Group0) with dispersed drug resistance information, so that whether the strain to be detected belongs to the drug resistant strain can be efficiently and accurately determined according to the base information of the SNP locus of the strain to be detected. The two Group-specific site combinations are shown in table 1 in the order 218514, 4907417, 5141114.
TABLE 1
According to an embodiment of the present invention, the SNP sites further include at least one SNP site selected from the group listed in table 2. For example, when the base at 49614 is T, 100% is clade 1; when the base of the site is C, 100 percent of the base is clade 2; when the base at this position is A, 100% of the DNA fragments are clade 3. According to the base condition of the site, one of three clades belonging to different drug-resistant clusters can be judged, and the other 25 sites are analogized in turn. The drug-resistant cluster can be distinguished by 100% according to the base condition of any one of the 26 sites.
TABLE 2
According to the embodiment of the invention, aiming at the systematic evolution relationship and drug sensitive phenotype results of 2676 Klebsiella pneumoniae, the inventor finds a combination of 3 sites to distinguish large clusters with drug resistance relationship (Table 1). The large clusters are divided into Group1 with high drug resistance rate and concentrated distribution of drug resistant samples and Group0 with low drug resistance rate and dispersed distribution of drug resistant samples.
According to the embodiment of the invention, the inventor further divides the sample in Group1 into 3 branches of clade1, clade2 and clade3 with drug resistance characteristics according to the system evolution relation and the drug sensitivity detection result. Based on this, some SNP molecular markers that can specifically distinguish three clades were found from the sample genome (Table 2).
According to the specific embodiment of the invention, a system evolution relation is constructed: the invention is based on 2676 Klebsiella pneumoniae evolution branch KP1 samples with the strongest drug resistance collected from 2013 to 2018 in a drug-resistant bacteria bank of a hospital in China. Firstly, whole genome sequencing is carried out on all samples, SNP (single nucleotide polymorphism) trapping is carried out, and the system evolution relation is constructed according to the obtained SNP matrix. Acquisition of drug-resistant and sensitive phenotype of the strain: drug sensitive phenotype experiments were performed on all samples for 11 drugs including Acamicin (AK), Aztreonam (ATM), Cefoperazone (CFP), Ciprofloxacin (CIP), Ceftriaxone (CRO), Ertapenem (ETP), Gentamicin (GEN), Imipenem (IPM), Levofloxacin (LVX), sulfamethoxazole-trimethoprim (SXT), Tobramycin (TOB), identifying resistant and sensitive phenotypes of the strains. Combining the system evolution relationship and the drug resistance information of the sample, screening large clusters with obvious drug resistance characteristics, and finding a combination consisting of 3 sites to distinguish the large clusters with the drug resistance relationship (Table 1). The large clusters are divided into a Group1 with drug resistance information distributed in a centralized manner and a Group0 with drug resistance information distributed in a dispersed manner (fig. 1). According to the guidelines of the american society for Infectious Diseases (IDSA), if a drug resistance rate of a certain species to a certain drug is higher than 20%, the drug is not recommended for the treatment of diseases caused by the species. Based on the principle, the samples in Group1 can be divided into 3 branches of clade1, clade2 and clade3 with drug resistance characteristics according to the system evolution relationship and the drug sensitivity detection result. Based on this, some SNP molecular markers capable of specifically distinguishing three clades are found from the sample genome. The screening mode is as follows: based on the SNP table obtained by SNP calling, a specific site which can strictly distinguish three clades is screened out through a python script. For example, the site is 100% of one base in clade1, 100% of another base in clade2, and 100% of another base in clade 3. The inventor screens 26 important SNP sites together to form the SNP molecular marker for distinguishing the drug-resistant clusters (figure 2). Identification of 3 branch-specific SNP markers: and traversing the whole genome SNP sites of all samples in Group1, and screening out a marker site which can specifically distinguish each clade from other two clades. That is, at this site, 100% of samples in clade1 were a certain base, 100% were another base in clade2, and 100% were another base in clade 3.
In a second aspect of the invention, there is provided a method of determining the drug sensitive phenotype of klebsiella pneumoniae, comprising: (1) determining 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, 218514 th, 4907417 th and 5141114 th; 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 Klebsiella pneumoniae to be detected belongs to a drug-resistant strain can be rapidly and effectively identified, and further the strain can be further analyzed and researched or a more accurate medication scheme can be made.
According to the embodiment of the invention, the method can be used for drug screening, for example, the method is used for determining the drug resistance of the strain and the drug resistance rate of the strain to a certain type of antibiotic, and whether a new drug to be detected is effective to a specific strain of Klebsiella pneumoniae is predicted based on the drug resistance mechanism of microorganisms to the specific type of antibiotic.
According to the embodiment of the invention, when the bases of the 218514, 4907417 and 5141114 sites are T A T in sequence, the resistance of the strain to be tested is indicated.
According to the embodiment of the invention, when the bases of the 218514, 4907417 and 5141114 sites are sequentially C C C, the base is an indication that the strain to be tested does not have drug resistance.
According to the embodiment of the invention, when the bases of the 218514, 4907417 and 5141114 sites are sequentially Tc, the resistance of the strain to be tested is indicated.
According to the embodiment of the invention, when the bases of the 218514, 4907417 and 5141114 sites are sequentially ccT, the indication that the strain to be tested does not have drug resistance is given.
According to the embodiment of the invention, when the bases at the 218514, 4907417 and 5141114 sites are sequentially C AC, the base sequence is an indication that the strain to be tested does not have drug resistance.
According to the embodiment of the invention, when the bases of the 218514, 4907417 and 5141114 sites are sequentially T C T, the T C T is an indication that the strain to be tested does not have drug resistance.
According to the embodiment of the invention, when the bases of the 218514, 4907417 and 5141114 sites are sequentially C AT, the base is an indication that the strain to be tested does not have drug resistance.
According to the embodiment of the invention, when the bases of the 218514, 4907417 and 5141114 sites are sequentially T AC, the base is an indication that the strain to be tested does not have drug resistance.
According to the embodiment of the present invention, when the test strain is judged to have drug resistance according to the above criteria, it is classified as Group1, and when the test strain is judged to have no drug resistance according to the above criteria, it is classified as Group 0.
According to the embodiment of the invention, the Group0 drug resistance rate is relatively low, drug resistant samples are scattered in the system evolution relationship, and when the strain to be tested is judged as Group0, traditional drug sensitive experiments can be carried out to determine the drug resistance.
According to the embodiment of the invention, when the strain to be tested is judged as Group1, the clade1, clade2 and clade3 can be further determined according to the base types of the SNP sites shown in the table 2. According to the embodiment of the invention, when the base of the 49614 site is T, the strain sample is classified into clade 1; when the base of the site is C, the strain sample is classified into clade 2; when the base at this site is A, the strain sample is classified as clade 3.
According to the embodiment of the invention, any site or any combination of sites in table 2 can be used for judging the assignment of the clade of the sample to be detected, and the clade is judged to be clade1, clade2 or clade 3. When the base at the 4839205 site is T, the strain sample is classified into clade 1; when the base of the site is C, the strain sample is classified into clade 2; when the base at this site is A, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 155494 site is A, the strain sample is classified into clade 1; when the base of the site is C, the strain sample is classified into clade 2; when the base at this site is T, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 283083 site is T, the strain sample is classified into clade 1; when the base of the site is G, the strain sample is classified into clade 2; when the base at this site is C, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 449376 site is T, the strain sample is classified into clade 1; when the base of the site is C, the strain sample is classified into clade 2; when the base at this site is G, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 495369 site is A, the strain sample is classified into clade 1; when the base of the site is C, the strain sample is classified into clade 2; when the base at this site is T, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 573182 site is T, the strain sample is classified into clade 1; when the base of the site is C, the strain sample is classified into clade 2; when the base at this site is A, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base at the 573299 site is G, the strain sample is classified into clade 1; when the base of the site is C, the strain sample is classified into clade 2; when the base at this site is T, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 577551 site is A, the strain sample is classified into clade 1; when the base of the site is C, the strain sample is classified into clade 2; when the base at this site is T, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base at the 775891 site is C, the strain sample is classified into clade 1; when the base of the site is G, the strain sample is classified into clade 2; when the base at this site is A, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base at the 1075460 site is A, the strain sample is classified into clade 1; when the base of the site is G, the strain sample is classified into clade 2; when the base at this site is T, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 1116497 site is T, the strain sample is classified into clade 1; when the base of the site is C, the strain sample is classified into clade 2; when the base at this site is A, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 1177312 site is T, the strain sample is classified into clade 1; when the base of the site is C, the strain sample is classified into clade 2; when the base at this site is G, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 1222291 site is C, the strain sample is classified into clade 1; when the base of the site is T, the strain sample is classified into clade 2; when the base at this site is G, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 1284861 site is T, the strain sample is classified into clade 1; when the base of the site is A, the strain sample is classified into clade 2; when the base at this site is G, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 1580566 site is A, the strain sample is classified into clade 1; when the base of the site is G, the strain sample is classified into clade 2; when the base at this site is T, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 1662442 site is C, the strain sample is classified into clade 1; when the base of the site is A, the strain sample is classified into clade 2; when the base at this site is T, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 2055657 site is C, the strain sample is classified into clade 1; when the base of the site is T, the strain sample is classified into clade 2; when the base at this site is G, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base at the 3989835 site is G, the strain sample is classified into clade 1; when the base of the site is C, the strain sample is classified into clade 2; when the base at this site is A, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base at the 4088637 site is G, the strain sample is classified into clade 1; when the base of the site is C, the strain sample is classified into clade 2; when the base at this site is A, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base at the 4150905 site is G, the strain sample is classified into clade 1; when the base of the site is C, the strain sample is classified into clade 2; when the base at this site is T, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base at the 4494702 site is G, the strain sample is classified into clade 1; when the base of the site is A, the strain sample is classified into clade 2; when the base at this site is C, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base at the 5101801 site is G, the strain sample is classified into clade 1; when the base of the site is T, the strain sample is classified into clade 2; when the base at this site is A, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 5123449 site is C, the strain sample is classified into clade 1; when the base of the site is T, the strain sample is classified into clade 2; when the base at this site is G, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 5128843 site is C, the strain sample is classified into clade 1; when the base of the site is G, the strain sample is classified into clade 2; when the base at this site is A, the strain sample is classified as clade 3.
According to the embodiment of the invention, when the base of the 5154735 site is A, the strain sample is classified into clade 1; when the base of the site is T, the strain sample is classified into clade 2; when the base at this site is C, the strain sample is classified as clade 3.
It should be noted that, when constructing the classification, the inventors classify the sample in Group1 into 3 branches clade1, clade2 and clade3 with drug-resistant characteristics according to the system evolution relationship and the drug-sensitive detection result, and the SNP sites and their corresponding bases shown in table 2 are SNP molecular markers specific for distinguishing the three branches.
According to an embodiment of the present invention, after the step (2), the method further comprises performing a drug resistance rate judgment on the test strain based on table 2. The resistance of the test strain to 11 drugs can be judged according to the classification of clade, as shown in Table 3.
Table 3: drug resistance per clade for each drug
According to the examples of the present invention, the strains classified as clade1 were high in drug resistance, with an average drug resistance of about 85%, the strains classified as clade2 were low in drug resistance, with an average drug resistance of about 5%, and the strains classified as clade3 were the second highest in drug resistance, with an average drug resistance of about 60%. The specific drug resistance rate of each drug still needs to be determined one by one according to the table three. In a third aspect, the present invention provides a kit for determining the drug sensitive phenotype of klebsiella pneumoniae, characterized in that it comprises 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: taking Klebsiella pneumoniae genome HS11286 as a reference genome, 218514 th, 4907417 th and 5141114 th. 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 the branch where the strain to be detected is located 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 includes a probe that can recognize at least one of the SNP sites listed in Table 2.
According to the embodiment of the invention, the probe can further comprise a fluorescent group, the specific probe can emit fluorescence when combined with a specific SNP locus, and the base type of the SNP locus of the strain to be detected is further determined, so that the drug resistance information of the strain to be detected is judged. The probe can also be used for target capture sequencing, specifically acquiring the sequence information of the SNP locus of the strain to be detected, and further judging the drug resistance information of the strain to be detected.
According to an embodiment of the present invention, the kit further comprises a primer that can amplify at least one of the SNP sites listed in Table 2. The primer can specifically amplify the SNP locus, and sequence information of the SNP locus is obtained by a sequencing technology, so that the drug resistance information of the strain to be detected is judged.
It should be noted that the "sequencing" or "sequencing technology" mentioned 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, next generation sequencing, single molecule sequencing, etc.
In a fourth aspect of the invention, an assay device for determining the drug sensitive phenotype of klebsiella pneumoniae is presented. According to an embodiment of the present invention, as illustrated in fig. 3, the analysis apparatus includes:
a SNP site determination unit 100 determining 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, 218514 th, 4907417 th and 5141114 th; and a drug sensitive phenotype determining unit 200, wherein the drug sensitive phenotype determining unit 200 is connected with the SNP locus determining unit 100, and determines the drug sensitive phenotype of the strain to be detected based on the base type.
According to an embodiment of the present invention, as shown in fig. 3, the apparatus further comprises: the drug resistance rate determination unit 300 determines the drug resistance rate of 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 of the second aspect of the present invention, and the biological principles thereof are the same as those of the method of the second aspect of the present invention, and will not be described herein again.
In a fifth aspect of the invention, the invention proposes a device for guiding medication for determining a medication for the treatment of a klebsiella pneumoniae related disease. According to an embodiment of the present invention, as shown in fig. 4, the apparatus includes: the analysis apparatus 1000 according to the fourth aspect of the present invention; and a drug determination device 2000, the drug determination device 2000 being connected to the analysis device 1000, the drug determination device determining a drug for treating klebsiella pneumoniae based on a result of the analysis device.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
Examples
(1) After a Klebsiella pneumoniae sample to be detected is obtained, whole genome sequencing is carried out on the Klebsiella pneumoniae sample, and SNP trapping is carried out by taking Klebsiella pneumoniae genome HS11286(Genebank access: NC-016845) as a reference.
(2) Based on the results of SNP calling, Group1, which had a high drug resistance rate and was distributed in a concentrated manner in drug-resistant samples, and Group0, which had a low drug resistance rate and was distributed in a dispersed manner in drug-resistant samples, were discriminated on the basis of the 3 sites (218514, 4907417, 5141114) in the reference sequence HS11286, and the combination of the 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 order of sites (218514, 4907417, 5141114) in table 1. For example: if the genome of the sample is T, A, T on positions 218514, 4907417, 5141114 in order, the sample can be classified as Group 1. Because Group0 has a relatively low drug resistance rate and the drug-resistant samples are scattered in the phylogenetic relationship, it is recommended that the samples classified as Group0 be subjected to traditional drug susceptibility testing to determine their drug resistance. Samples classified as Group1 can then continue to be classified according to SNP molecular markers.
(3) In the samples of Group1, Group1 could be divided into 3 clades based on the following positions on the reference sequence HS 11286. As shown in table 2. For example: if a sample that has been judged to be Group1 is "T" at position 49614 or meets the specific SNP molecular markers of other clades 1 in Table 2, the sample can be classified as clade 1. Clade2 and Clade3 and so on.
(4) The drug resistance of the sample to 11 drugs can be judged according to the classification of the clade. The sample resistance is summarized in Table 3.
According to the guidelines of the american society for Infectious Diseases (IDSA), antibiotics cannot be used to treat diseases caused by a certain species if the resistance rate of the antibiotic to the species is greater than 20%. Based on the statistical results of 1064 samples, the drug resistance of the strain clade1 was high, with an average drug resistance of about 85%, the drug resistance of the strain classified as clade2 was low, with an average drug resistance of about 5%, and the drug resistance of the strain classified as clade3 was the second highest, with an average drug resistance of about 60%.
Depending on the application scenario, if a drug resistance determination is required for a certain drug, it can be further analyzed based on table 3. For example: for AK (amikacin), the resistance rate of clade2 and 3 is low, classified as sensitivity, and the resistance rate of clade1 as the clade with the highest resistance degree shown by a resistance spectrum is lower than that of other drugs; for ATM (aztreonam), only clade2 is sensitive, clade3 exceeds a drug resistance threshold and is classified as drug resistance, the drug resistance rate of clade1 exceeds 90%, and the drug resistance rate is extremely high; for SXT (sulfamethoxazole-trimethoprim), clade2 still showed sensitivity, and both clade1 and clade3 showed some degree of drug resistance.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean 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 invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer 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, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (11)
1. Use of a reagent for detecting an SNP site for the preparation of a kit for determining the drug sensitive phenotype of Klebsiella pneumoniae, wherein the SNP site comprises at least one of: taking Klebsiella pneumoniae genome HS11286 as a reference genome, 218514 th, 4907417 th and 5141114 th.
2. The use of claim 1, wherein said SNP sites further include at least one SNP site selected from those listed in table 2.
3. A method of determining the 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, 218514 th, 4907417 th and 5141114 th; and
(2) and determining the drug sensitive phenotype of the strain to be detected based on the base type.
4. The method of claim 3, wherein the bases at positions 218514, 4907417 and 5141114 are TA T in sequence, which is indicative of drug resistance of the test strain;
optionally, when the bases at the 218514, 4907417 and 5141114 sites are sequentially C C C C, the base is an indication that the strain to be tested does not have drug resistance;
optionally, when the bases at the 218514, 4907417 and 5141114 sites are sequentially Tc, the resistance is indicated that the strain to be tested does not have resistance;
optionally, when the bases at the 218514, 4907417 and 5141114 sites are sequentially cCTs, the base is an indication that the strain to be tested does not have drug resistance;
optionally, when the bases at the 218514, 4907417 and 5141114 sites are sequentially C A C, the base is an indication that the strain to be tested does not have drug resistance;
optionally, when the bases at the 218514, 4907417 and 5141114 sites are sequentially T C T, the determination is an indication that the strain to be tested does not have drug resistance;
optionally, when the bases at the 218514, 4907417 and 5141114 sites are sequentially cAT, the indication that the strain to be tested does not have drug resistance is given;
optionally, when the bases at the 218514, 4907417 and 5141114 sites are sequentially tAC, the base is an indicator that the strain to be tested does not have drug resistance.
5. The method of claim 3, wherein said SNP sites further comprise at least one SNP site selected from the group consisting of the SNP sites set forth in Table 2.
6. The method according to claim 3, wherein after the step (2), the method further comprises the step of judging the drug resistance of the test strain based on Table 2.
7. A kit for determining the drug sensitive phenotype of klebsiella pneumoniae comprising primers and/or probes for identifying and/or amplifying at least one of the following SNP sites: taking Klebsiella pneumoniae genome HS11286 as a reference genome, 218514 th, 4907417 th and 5141114 th.
8. The kit of claim 7, further comprising a probe that recognizes at least one of the SNP sites listed in Table 2;
optionally, primers that can amplify at least one of the SNP sites listed in Table 2 are further included.
9. An assay device for determining the drug sensitive phenotype of klebsiella pneumoniae comprising:
an SNP site determination unit determining 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, 218514 th, 4907417 th and 5141114 th;
and a drug sensitive phenotype determination unit which determines the drug sensitive phenotype of the strain to be tested based on the base type.
10. The apparatus of claim 9, further comprising:
and a drug resistance rate determination unit which judges the drug resistance rate of the strain to be tested based on the table 2.
11. An apparatus for guiding medication, the apparatus for determining a medication for treating a klebsiella pneumoniae-related disease, comprising:
the assay device of claim 9 or 10; 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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