CN112760393A

CN112760393A - Urinary system infection pathogen drug-resistant gene detection system and kit and application thereof

Info

Publication number: CN112760393A
Application number: CN202110176576.3A
Authority: CN
Inventors: 张艳梅; 赵虎; 孙召洋; 吴勇; 南丽; 余丁
Original assignee: Ningbo Health Gene Technologies Co ltd; Huadong Hospital
Current assignee: Ningbo Health Gene Technologies Co ltd; Huadong Hospital
Priority date: 2021-02-07
Filing date: 2021-02-07
Publication date: 2021-05-07
Anticipated expiration: 2041-02-07
Also published as: CN112760393B

Abstract

The invention relates to a detection system for drug-resistant genes of urinary system infection pathogens, a kit and application thereof, wherein the detection system comprises 10 pairs of primers, wherein the detection system comprises 9 pairs of drug-resistant gene primers and 1 pair of system quality control reference primers. The urinary infection pathogen resistance genes are directed against ESBLs, carbapenemase, MRSA and VRE genotypes, respectively. The urinary system infection pathogen drug-resistant gene detection system, the kit and the application thereof do not need to adopt conventional detection, can directly carry out synchronous detection and analysis on multiple pathogen drug-resistant genes on a urine sample in the same reaction system, make up for the defects of low flux, long time consumption and the like of the conventional detection method, provide comprehensive, accurate and low-cost etiology diagnosis for clinic in the first time, and provide important references for individual medication and accurate medical treatment.

Description

Urinary system infection pathogen drug-resistant gene detection system and kit and application thereof

Technical Field

The invention relates to a multiple gene detection product and a detection system used by the product, belonging to the technical field of biology.

Background

Urinary Tract Infection (UTI), also known as Urinary Tract infection, is one of the most common clinical infectious diseases, second to respiratory Tract infection in outpatient service. The pathogens causing the UTI are various, and the pathogens frequently separated from the urine sample are Escherichia coli, Klebsiella pneumoniae, enterococcus faecalis, enterococcus faecium, Pseudomonas aeruginosa, Candida albicans, Proteus mirabilis, Streptococcus agalactiae and the like in sequence. The most effective treatment for urinary tract infection is antibiotic treatment, but with the widespread and overuse of antibiotics, the resistance of urinary tract infection pathogens to common antibiotics is gradually increased, and the problem of the resistance of urinary tract infection common bacteria to antibiotics is quite common in people. Although the pathogens and drug resistance of urinary tract infection of different countries, regions and groups are different, the problem of bacterial drug resistance has gradually become a big problem and threat of public health all over the world. In addition, antibiotic resistance is one of the ten major threats facing the global health in 2019 released by WHO, and its seriousness and harmfulness are increasing.

Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis are reported to have increasingly severe resistance to commonly used antibiotics such as β -lactams and carbapenems; there are increasing numbers of escherichia coli, klebsiella pneumoniae, and proteus mirabilis producing extended spectrum beta-lactamase (ESBLs), and many reports indicate that the average incidence of ESBLs production by escherichia coli and klebsiella pneumoniae is close to 50%. In the last decade, carbapenem-resistant Enterobacteriaceae (CRE) bacteria such as escherichia coli and klebsiella pneumoniae appear and spread rapidly in the world, which brings great trouble to the medication of clinicians, and how to rapidly and accurately detect the CRE strain becomes a hotspot of antibacterial treatment. Research shows that the drug resistance mechanism of CRE mainly comprises the following three aspects: carbapenemase, including KPC enzyme in A enzyme; VIM and IMP in B enzymes, and superbacteria carrying NDM-1 type enzymes have also been reported; OXA enzymes among class D enzymes; deletion of porin and/or high expression of cephalosporins; hyperfunction of an active efflux pump and change of action targets of carbapenem antibacterial drugs on bacteria. Among these resistance mechanisms, the most important is the production of carbapenemases, and the most important are the class a and B enzymes. Carbapenemases are a class of beta-lactamases that hydrolyze carbapenem antibiotics and can be classified into A, B, D types based on carbapenemases, wherein A, D belongs to serinases (including KPC, IMI, NMC-A, SME, etc.) and B belongs to metalloenzymes (including NDM, IMP, VIM, SIM, etc.). From the present point of view, KPC is the most prevalent carbapenemase type on a global scale. CRE has become an important clinical challenge at present, and has fast spread and high fatality rate. KPC and NDM are the most important drug resistance mechanism, can be transmitted by plasmid, and early detection and monitoring are important measures for avoiding CRE transmission. After the Klebsiella pneumoniae which produces KPC enzyme is reported for the first time in the United states, the pathogen is widely detected in the world as a multi-drug resistant bacterium, and a serious challenge is brought to the anti-infection work in the world. Once lung produces KPC enzyme, carbapenems are hydrolyzed, and the carbapenems are the last line of defense for controlling gram-negative bacteria infection at present, and reports show that the final fatality rate can reach 75% due to the lack of effective drugs for controlling the infection caused by the lung producing KPC enzyme. Therefore, the rapid determination of whether the bacteria carry the carbapenemase gene or not through molecular detection has important significance for monitoring the drug resistance of the bacteria and preventing the spread and transmission of drug-resistant bacteria. In addition, because the different carbapenemases have certain differences in the types of medicines used, the rapid determination of the carbapenemase genotype is helpful to provide certain medicine guide for clinicians.

As the problem of drug resistance of bacteria becomes more serious, the simultaneous detection of pathogen and drug resistance genes becomes more and more important. CLSI100 describes the detection of drug resistance genes in great detail and is also recommended. For example, the MacA gene, is recommended to consider molecular target technology for detection, which is more accurate than that of some phenotypic experiments. In the case of detecting Staphylococcus aureus, the MecA/C gene and MREJ fragment, which are drug resistance genes, have been reported. Negative Predictive Value (NPV) for MRSA (methicillin-resistant staphylococcus aureus) is 100%; can assist in reducing the overuse of antibiotics such as vancomycin and the like, thereby restraining the drug resistance trend of bacteria to a certain extent.

Because the clinical manifestations caused by bacterial infection often present non-specificity and are accompanied by a plurality of complications, the specificity of auxiliary diagnosis is low, and the clinician faces to have a certain diagnosis and treatment predicament when suffering from infectious diseases, resulting in low diagnosis accuracy. According to the related report, the following results are shown: 35% doctors usually take medicines according to experience, 47% doctors take medicines according to culture, 5% doctors take medicines according to requirements of patients, and a laboratory is clinically required to give accurate pathogeny detection and drug sensitivity test report results as soon as possible, however, as the traditional culture method takes 3-5 days on average, bacteria and fungi which are difficult to culture need more than one week, the culture positive rate is low, and the drug sensitivity test needs a single colony separated from a flat plate to be carried out, so that the goals of rapidly detecting pathogens and determining the drug resistance of the pathogens in the laboratory are difficult to realize.

In addition, the treatment of urinary tract infections caused by different pathogens varies from one pathogen to another. The clinical application guideline of antibacterial drugs in 2015 of national committee of health clearly indicates that: "the application of the antibacterial agent must be clearly applied after diagnosis according to the symptoms, signs and laboratory examination results of the patient". However, the conventional detection method at present has the defects of low detection rate, long time consumption, especially incapability of accurately identifying multiple pathogens at the same time and the like, and cannot provide timely, comprehensive and accurate pathogen diagnosis basis for clinic, so that the problems of low curative effect, incapability of controlling urinary tract infection progress in time, induction of high drug resistance rate of bacterial strains, increase of medical burden of patients and the like caused by empirical treatment by adopting broad-spectrum antibacterial drugs in clinic are caused.

In summary, in order to make clear the types of drug-resistant genes of common pathogens of urinary system infection as early as possible, continuously perform drug-resistant monitoring work, fully understand the types of bacteria and the changes of the drug-resistant rates of antibacterial drugs, better guide the clinical reasonable medication, and urgently need to develop a new technology.

Disclosure of Invention

The invention aims to provide a rapid, comprehensive, accurate and low-cost detection system and kit for drug-resistant genes of urinary system infection pathogens, and application of the detection system in preparation of diagnostic products.

The invention provides a technical scheme for solving the technical problems, which comprises the following steps: a urinary system infection pathogen drug-resistant gene detection system comprises forward and reverse primers for detecting blaCTX-M gene of ultra-broad spectrum beta-lactamase, forward and reverse primers for detecting blaSVV gene of ultra-broad spectrum beta-lactamase, forward and reverse primers for detecting blaKPC gene of carbapenemase, forward and reverse primers for detecting blaNDM gene of carbapenemase, forward and reverse primers for detecting blaIMP gene of carbapenemase, forward and reverse primers for detecting blaVIM gene of carbapenemase, forward and reverse primers for detecting mecA gene of methicillin-resistant staphylococcus aureus, forward and reverse primers for detecting mecC gene of methicillin-resistant staphylococcus aureus, and forward and reverse primers for detecting vanA gene of vancomycin-resistant enterococcus, the test sample is urine.

The nucleotide sequence of the forward primer aiming at blaCTX-M is shown as SEQ ID No.1, and the nucleotide sequence of the reverse primer aiming at blaCTX-M is shown as SEQ ID No. 2;

the nucleotide sequence of the forward primer aiming at the blaSVV is shown as SEQ ID No.3, and the nucleotide sequence of the reverse primer aiming at the blaSVV is shown as SEQ ID No. 4;

the nucleotide sequence of the forward primer aiming at blaKPC is shown as SEQ ID No.5, and the nucleotide sequence of the reverse primer aiming at blaKPC is shown as SEQ ID No. 6;

the nucleotide sequence of the forward primer aiming at the blaNDM is shown as SEQ ID No.7, and the nucleotide sequence of the reverse primer aiming at the blaNDM is shown as SEQ ID No. 8;

the nucleotide sequence of the forward primer for blaIMP is shown as SEQ ID No.9, and the nucleotide sequence of the reverse primer for blaIMP is shown as SEQ ID No. 10;

the nucleotide sequence of the forward primer for blaVIM is shown as SEQ ID No.11, and the nucleotide sequence of the reverse primer for blaVIM is shown as SEQ ID No. 12;

the nucleotide sequence of the forward primer aiming at mecA is shown as SEQ ID No.13, and the nucleotide sequence of the reverse primer aiming at mecA is shown as SEQ ID No. 14;

the nucleotide sequence of the forward primer aiming at the mecC is shown as SEQ ID No.15, and the nucleotide sequence of the reverse primer aiming at the mecC is shown as SEQ ID No. 16;

the nucleotide sequence of the forward primer for vanA is shown as SEQ ID No.17, and the nucleotide sequence of the reverse primer for vanA is shown as SEQ ID No. 18.

The detection system of the drug-resistant gene of the urinary system infection pathogen also comprises forward and reverse primers for detecting the quality control internal reference of the system; the nucleotide sequence of the forward primer aiming at the system quality control internal reference is shown as SEQ ID No.19, and the nucleotide sequence of the reverse primer aiming at the system quality control internal reference is shown as SEQ ID No. 20.

The final concentration of all the forward primers in the detection system is 200 nM; the final concentration of all reverse primers in the detection system was 200 nM.

The detection system for the drug-resistant genes of the urinary system infection pathogens further comprises multiple PCR premixed liquid, multiple PCR enzyme liquid and nuclease-free pure water; the multiplex PCR premix consists of 10 XPCR buffer solution and MgCl₂Mixing with dNTPs; the multiple PCR enzyme solution is formed by mixing hot start DNA polymerase and UNG enzyme.

And the 5' end of each forward primer is provided with a fluorescent label which is CY5 or CY3 or FAM.

The urinary system infection pathogen drug-resistant gene detection system also comprises a positive control substance and a negative control substance; the positive control is a plasmid mixture comprising all target gene targets; the negative control was nuclease-free ultrapure water.

The components used in the reaction system are 10 XPCR buffer solution 1 volume, 10. mu.M dNTPs 0.2 volume, 25mmol/L MgCl₂0.8 volume of the solution, 1 volume of the primer mixture, 0.4 volume of 5U/. mu.L of hot start DNA polymerase, 0.5 volume of 1U/. mu.L of UNG enzyme, 5 volumes of DNA template, and 1.1 volume of nuclease-free pure water; the using amount of the gene template is 5-50 ng/system.

The invention provides another technical scheme for solving the technical problems, which comprises the following steps: a kit for detecting the drug-resistant gene of the pathogen of urinary system infection, which comprises the detection system.

The invention provides another technical scheme for solving the technical problems, which comprises the following steps: an application of the detection system in preparing the drug-resistant gene detection and diagnosis product of urinary system infection pathogens.

The invention has the positive effects that:

(1) the product for detecting the drug resistance genes of the urinary system infection pathogens can simultaneously identify a plurality of drug resistance genes of the urinary system infection pathogens through urine, mix the copy numbers of plasmids and the like of all target genes together, and adjust the primer concentration of each pathogen to ensure that the peak heights of all target spots are equivalent, thereby achieving the purpose of equivalently amplifying all target genes. The detection product can detect urine samples of patients with urinary system infection or suspected patients, provides the drug resistance genotype of pathogens of urinary system infection, helps clinicians to define the drug resistance genotype of the pathogens in time, reduces the use of empirical antibiotics to a certain extent, is helpful for monitoring the transmission of important drug resistance genes, prevents the transmission and the prevalence of the drug resistance genes in hospitals, and reduces the medical cost.

(2) The detection product of the drug-resistant gene of the urinary system infectious pathogen is added with the UNG enzyme for preventing pollution, effectively eliminates the pollution of gene amplification fragments before gene amplification, and ensures the accuracy and reliability of results.

(3) The product for detecting the drug resistance gene of the urinary system infection pathogen is different from the traditional gel electrophoresis analysis mode, and can separate a non-specific amplification product, a primer dimer and a specific amplification product, so that the detection result has no foreign peak, and the specificity and the sensitivity of the detection are ensured. Tests prove that the method has fewer peaks and shows high specificity; and can detect drug-resistant genes with the copy number as low as 10/mu L, and has higher sensitivity.

(4) The product for detecting the drug-resistant gene of the urinary system infection pathogen synchronously adds the IC internal reference, thereby ensuring the detection accuracy. The IC can monitor the reaction processes of extraction, PCR and capillary electrophoresis, and the failure of reaction can be indicated when no IC characteristic peak appears, so that false negative can be effectively avoided.

(5) The regimen and treatment of urinary infections caused by different pathogens varies. The clinical application guideline of antibacterial drugs in 2015 of national committee of health clearly indicates that: "the application of the antibacterial agent must be clearly applied after diagnosis according to the symptoms, signs and laboratory examination results of the patient". However, the conventional detection method at present has the defects of low detection rate, long time consumption, particularly incapability of accurately identifying multiple pathogens at the same time and the like, so that the problems of low curative effect, incapability of timely controlling urinary system infection, high drug-resistant strain rate, waste of national medical resources, increase of medical cost of patients and the like caused by empirical treatment by clinically and generally adopting broad-spectrum antibacterial drugs are caused. The invention establishes a high-throughput, rapid, accurate and low-cost identification system for drug-resistant genes of urinary system infection pathogens, can synchronously detect the drug-resistant genes of 9 urinary system infection pathogens, effectively overcomes the defects of low detection rate, long time consumption, incapability of simultaneously detecting the drug-resistant genes of various pathogens and the like of a conventional detection method, can determine the types and types of the drug-resistant genes of the pathogens within 2.5 hours, so that a correct treatment scheme and isolation measures can be clinically adopted, the infection diffusion is effectively prevented, and the generation of drug-resistant strains is reduced.

Drawings

FIG. 1 is a diagram of the kit of example 1 after performing a PCR reaction on a mixed positive control and performing capillary electrophoresis analysis;

FIG. 2 is a diagram of the kit of example 1 of the present invention after performing a PCR reaction on a negative control and performing capillary electrophoresis analysis;

FIG. 3 is a diagram of a sample 1 subjected to a PCR reaction and then to capillary electrophoresis analysis using the kit of example 1 of the present invention;

FIG. 4 is a diagram of a sample 2 after a PCR reaction and a capillary electrophoresis analysis by the kit of example 1 of the present invention;

FIG. 5 is a diagram of a sample 3 after a PCR reaction and a capillary electrophoresis analysis by the kit of example 1 of the present invention;

FIG. 6 is a diagram showing a sample 4 subjected to a PCR reaction and then to capillary electrophoresis analysis by the kit of example 1 of the present invention.

Detailed Description

The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-described disclosure. In the following examples, the reagents used were all analytical grade and were commercially available unless otherwise indicated. Experimental procedures not specifically identified herein are generally carried out under conventional conditions such as those described in the molecular cloning guidelines, published by scientific Press 2002, edited by J. SammBruk et al, or under conditions recommended by the manufacturer. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.

Example 1

Composition of kit

The kit for detecting the drug-resistant gene of the pathogen infecting the urinary system comprises: multiplex PCR premix, multiplex PCR enzyme solution, and primer mixturePositive control, negative control, system quality control internal reference (IC) and nuclease-free pure water. The multiplex PCR premix consists of 10 XPCR buffer solution and MgCl₂And dNTPs. The multiple PCR enzyme solution is formed by mixing hot start DNA polymerase and UNG enzyme.

Both the multiplex PCR master mix and the multiplex PCR enzyme were obtained from Roche (cat. No.: 210212).

The positive control is a plasmid mixture that includes all of the gene targets of interest.

The negative control was nuclease-free ultrapure water.

The primer mixture comprises: the nucleotide sequence of the forward primer and the nucleotide sequence of the reverse primer for blaCTX-M; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for blasHV; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for blaKPC; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for blaNDM; the nucleotide sequence of the forward primer and the nucleotide sequence of the reverse primer for blaIMP; the nucleotide sequence of the forward primer and the nucleotide sequence of the reverse primer for blaVIM; the nucleotide sequence of the forward primer and the nucleotide sequence of the reverse primer for mecA; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for mecC; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for vanA; the nucleotide sequence of the forward primer and the nucleotide sequence of the reverse primer for the system quality control Internal Control (IC).

The sequences of the primers are characterized as shown in Table 1, and the primers were synthesized by Shanghai Biotech engineering Co., Ltd.

TABLE 1 primer sequence characterization Table

Second, using method of kit

The specific detection steps of the kit for detecting the drug-resistant genes of the urinary system infection pathogens are as follows:

(1) collecting a urine sample of a patient: clean midstream urine is collected from a patient suspected or diagnosed with urinary system infection.

(2) Extracting nucleic acid of a sample: A300-mu-L clean middle section urine sample is taken for nucleic acid extraction, 80 mu-L of each of the positive control and the negative control is taken for extraction, and each sample involved in extraction needs to be added with 3 mu-L of IC for extraction.

(3) Preparing a reaction system: according to the instruction, a reaction system is prepared according to the proportion of 2 mu L of each reaction multiple PCR premix, 0.9 mu L of multiple PCR enzyme solution, 1 mu L of primer mixture and 1.1 mu L of nuclease-free pure water, the reaction system is evenly mixed by vortex, and then is centrifuged by a centrifuge and is subpackaged in PCR reaction tubes.

(4) Adding a nucleic acid template: adding the extracted nucleic acid into a PCR reaction tube provided with a prepared reaction system, and adding 5 mu L of nucleic acid into each part.

(5) Multiplex PCR amplification: the PCR amplification reaction conditions of the kit are shown in Table 2.

TABLE 2 multiplex PCR amplification conditions

(6) And carrying out capillary electrophoresis analysis on the amplification products, and carrying out result interpretation according to a peak pattern.

3500Dx genetic analyzer matched with highly deionized formamide (HiDi)8.75 mu L, SIZE-500Plus0.25 mu L is taken, mixed and added with PCR product 1 mu L for capillary electrophoresis separation of samples. And judging the type of the pathogen drug resistance gene according to the size of the peak position of the peak pattern diagram.

Thirdly, judging the detection result of the kit

1. Kit validity determination

The result judgment can be carried out when the following conditions are met:

1) negative control: only specific peaks of the internal reference of the system quality control are detected.

2) Positive control: one fluorescence signal was detected at each amplified fragment length and the fluorescence signal value was above 500.

2. And (3) judging the validity of the sample:

1) if the fluorescence signal value of the detected sample is at least one value higher than 32000, the sample is added in an excessive amount, and the capillary electrophoresis detection is recommended after the PCR product is properly diluted.

2) If the fluorescence signal values of the detected samples are all lower than 500, the sample addition amount is lower, and the PCR product addition amount or the PCR reaction cycle number can be properly increased; if the requirements are still not met, the sample is prepared again.

3. Criteria for determination of results

Identification of drug-resistant genes of urinary system infection pathogens

Corresponding peaks appear in the target fragment region of the system quality control internal reference and pathogen resistance genes, the fluorescence signal values are all higher than 500, and the infected pathogen can be judged to carry the corresponding resistance genes.

4. Example of result judgment

And determining the type of the drug-resistant gene of the infectious pathogen according to the position of each target peak in the map.

The kit of this example was used to perform PCR reactions on individual positive controls and capillary electrophoresis analysis was performed. The target fragment region of the gene blaCTX-M, blaSVV, blaKPC, blaNDM, blaIMP, blaVIM, mecA, mecC and vanA showed corresponding peaks in 9 drug-resistant genes. The results were very visual and the genes were all well amplified. Thus, each pair of primers can effectively amplify the corresponding target gene and has good specificity.

The spectrum of the mixture of all positive controls after PCR reaction using the kit of this example and capillary electrophoresis analysis is shown in FIG. 1. The target fragment region blaCTX-M, blaSVV, blaKPC, blaNDM, blaIMP, blaVIM, mecA, mecC and vanA of the gene and 10 detection targets of the system quality control internal reference all show corresponding peaks. The results were very visual and the genes were all well amplified. Thus, the primers are not interfered with each other, and all target genes can be effectively amplified at the same time.

The spectrum of the negative control subjected to PCR reaction by using the kit of the embodiment and analyzed by capillary electrophoresis is shown in FIG. 2, and the characteristic peak of the Internal Control (IC) of the system quality control does not have any characteristic peak of pathogens, and only has non-specific background fluorescence signals at the position less than 100 nt. The detection system has good specificity.

The kit using method of the embodiment is adopted to carry out serial dilution on the positive control of the drug resistance gene of the single urinary system infection pathogen, and capillary electrophoresis analysis is adopted after PCR reaction is carried out, so as to evaluate the sensitivity of the method for detecting the drug resistance gene. As shown in Table 3, the minimum sensitivity of the method for detecting 9 drug-resistant genes was 10 copies/. mu.L. The detection system has higher sensitivity for detecting the single infection of the drug-resistant gene of the urinary system infection pathogen.

TABLE 3 detection sensitivity of drug-resistant genes of urinary system infection pathogens

FIG. 3 shows a spectrum obtained by analyzing a sample 1 by capillary electrophoresis after PCR reaction using the kit of this example. The system quality control internal reference IC appears and the signal value is more than 500, and the target fragment region of blaKPC gene has corresponding peak and the signal value is more than 500. According to the result judgment standard, the pathogen infected by the patient is indicated to carry the blaKPC drug-resistant gene. The detection result is very intuitive.

FIG. 4 shows a chromatogram obtained by analyzing a sample 2 by capillary electrophoresis after PCR reaction using the kit of this example. The system quality control internal reference IC appears and the signal value is more than 500, and the target fragment region of the blaNDM gene appears corresponding peaks and the signal value is more than 500. According to the result judgment standard, the pathogen infected by the patient is indicated to carry the blaNDM drug resistance gene. The detection result is very intuitive.

FIG. 5 shows a spectrum obtained by analyzing a sample 3 by capillary electrophoresis after PCR reaction using the kit of this example. The system quality control internal reference IC appears and the signal value is more than 500, and the target fragment region of blaCTX-M and blaSVV genes has corresponding peaks and the signal value is more than 500. According to the result judgment standard, the pathogen infected by the patient is indicated to carry blaCTX-M and blaSVV gene drug resistance genes. The detection result is very intuitive.

FIG. 5 shows a spectrum obtained by analyzing a sample 4 by capillary electrophoresis after PCR reaction using the kit of this example. The system quality control internal reference IC appears and the signal value is more than 500, and the target fragment region of blaIMP gene has corresponding peak and the signal value is more than 500. According to the result judgment standard, the pathogen infected by the patient is shown to carry the blaIMP drug-resistant gene, and the detection result is very visual.

It should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And such obvious variations or modifications which fall within the spirit of the invention are intended to be covered by the scope of the present invention.

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Claims

1. A urinary system infection pathogen drug-resistant gene detection system is characterized in that: the kit comprises forward and reverse primers for detecting blaCTX-M genes of the extended-spectrum beta-lactamase, forward and reverse primers for detecting blaSVV genes of the extended-spectrum beta-lactamase, forward and reverse primers for detecting blaKPC genes of carbapenemase, forward and reverse primers for detecting blaNDM genes of carbapenemase, forward and reverse primers for detecting blaIMP genes of carbapenemase, forward and reverse primers for detecting blaVIM genes of carbapenemase, forward and reverse primers for detecting mecA genes of methicillin-resistant staphylococcus aureus, forward and reverse primers for detecting mecC genes of methicillin-resistant staphylococcus aureus and forward and reverse primers for detecting vanA genes of vancomycin-resistant enterococcus, and a detection sample is urine.

2. The system for detecting resistance genes of urinary infection pathogens according to claim 1, wherein: the nucleotide sequence of the forward primer aiming at blaCTX-M is shown as SEQ ID No.1, and the nucleotide sequence of the reverse primer aiming at blaCTX-M is shown as SEQ ID No. 2;

to is directed atblaIMPThe nucleotide sequence of the forward primer of (1) is shown as SEQ ID No.9, directed againstblaIMPThe nucleotide sequence of the reverse primer of (1) is shown in SEQ ID No. 10;

to is directed atblaVIMThe nucleotide sequence of the forward primer of (1) is shown as SEQ ID No.11, directed againstblaVIMThe nucleotide sequence of the reverse primer of (1) is shown in SEQ ID No. 12;

to is directed atmecAThe nucleotide sequence of the forward primer of (1) is shown as SEQ ID No.13, directed againstmecAThe nucleotide sequence of the reverse primer of (1) is shown in SEQ ID No. 14;

to is directed atmecCThe nucleotide sequence of the forward primer of (1) is shown as SEQ ID No.15, directed againstmecCThe nucleotide sequence of the reverse primer of (1) is shown in SEQ ID No. 16;

to is directed atvanAThe nucleotide sequence of the forward primer of (1) is shown as SEQ ID No.17, directed againstvanAThe nucleotide sequence of the reverse primer of (1) is shown in SEQ ID No. 18.

3. The system for detecting the resistance gene of the urinary infection pathogen according to claim 2, wherein: the system also comprises forward and reverse primers for detecting the system quality control internal reference; the nucleotide sequence of the forward primer aiming at the system quality control internal reference is shown as SEQ ID No.19, and the nucleotide sequence of the reverse primer aiming at the system quality control internal reference is shown as SEQ ID No. 20.

4. The system for detecting resistance genes of urinary infection pathogens according to claim 3, wherein: the final concentration of all the forward primers in the detection system is 200 nM; the final concentration of all reverse primers in the detection system was 200 nM.

5. The system for detecting resistance genes to urinary infection pathogens according to any one of claims 1 to 4, wherein: the kit also comprises multiple PCR premix solution, multiple PCR enzyme solution and nuclease-free pure water; the multiplex PCR premix consists of 10 XPCR buffer solution and MgCl₂Mixing with dNTPs; the multiple PCR enzyme solution is formed by mixing hot start DNA polymerase and UNG enzyme.

6. The system for detecting resistance genes to urinary infection pathogens according to any one of claims 1 to 4, wherein: and the 5' end of each forward primer is provided with a fluorescent label which is CY5 or CY3 or FAM.

7. The system for detecting resistance genes to urinary infection pathogens according to any one of claims 1 to 4, wherein: positive and negative controls are also included; the positive control is a plasmid mixture comprising all target gene targets; the negative control was nuclease-free ultrapure water.

8. The system for detecting resistance genes of urinary infection pathogens according to claim 5, wherein: the components used in the reaction system are 10 XPCR buffer solution 1 volume, 10. mu.M dNTPs 0.2 volume, 25mmol/L MgCl₂0.8 volume of the solution, 1 volume of the primer mixture, 0.4 volume of 5U/. mu.L of hot start DNA polymerase, 0.5 volume of 1U/. mu.L of UNG enzyme, 5 volumes of DNA template, and 1.1 volume of nuclease-free pure water; the using amount of the gene template is 5-50 ng/system.

9. A kit for detecting a resistance gene of a urinary infection pathogen comprising the detection system according to claim 1.

10. Use of the detection system according to claim 1 for the preparation of a product for the detection and diagnosis of resistance genes of pathogens of urinary infections.