Primer combination and kit for detecting urinary tract infection pathogenic microorganisms and drug-resistant genes and application

Abstract

The invention discloses a primer combination and a kit for detecting urinary tract infection pathogenic microorganisms and drug-resistant genes and application, and belongs to the technical field of pathogenic microorganism and drug-resistant gene detection. The primer combination and the kit have the innovative technical characteristics of multiple detectability, adoption of a fluorescence labeling technology, realization of multiple detections in one tube and the like, can quickly and accurately detect various pathogenic microorganisms and drug-resistant genes, remarkably improve the diagnostic efficiency of urinary tract infection, and guide accurate medication. The detection method is also combined with the comprehensive detection capability based on the qPCR technology and a later screening and judging system, so that the detection sensitivity and specificity are further improved, the false positive rate is reduced, and more powerful support is provided for clinical treatment. The implementation of the invention is helpful for improving the quality and efficiency of medical services and meeting clinical requirements.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection of pathogenic microorganisms and drug-resistant genes, and particularly relates to a primer combination, a kit and an application for detecting pathogenic microorganisms and drug-resistant genes in urinary tract infections. Background Art

[0002] Urinary tract infection (UTI) is defined microbiologically as an inflammatory response of the urothelium to microbial pathogens and is one of the most common bacterial infections. When a UTI is accompanied by a co-existing disease that confers a risk of acquisition or treatment failure, such as structural or functional abnormalities of the urogenital tract, or other underlying diseases, it is a complicated urinary tract infection (cUTI). Because the inducing factors of cUTI are sometimes difficult or impossible to remove, and it is often caused by drug-resistant bacterial infections, the recurrence rate and treatment difficulty are increased, and there is a high risk of treatment failure. Therefore, early identification of the infecting microorganism is a key step to ensure effective subsequent medical intervention. Clinically, the gold standard for the diagnosis of pathogenic bacteria is still the isolation and culture method, which is time-consuming, has poor sensitivity, and cannot quickly guide drug use. In addition, the types of pathogens causing the disease are complex and difficult to clearly identify, making it impossible to assist clinicians in promptly finding the pathogens infecting the patient and giving effective treatment. Quantitative real-time polymerase chain reaction (qPCR) is a powerful tool for analyzing and quantifying gene expression. qPCR has the advantages of simple operation, fast and convenient, high sensitivity, good repeatability, low contamination rate, etc., and has great potential in clinical etiological diagnosis.

[0003] Although the qPCR technology has significant advantages in the detection of urinary tract infections, it still faces some challenges in practical applications. Existing urinary tract infection detection technologies often can only detect a few common pathogenic microorganisms, such as Escherichia coli, Klebsiella pneumoniae, etc., and cannot effectively detect some uncommon or newly emerging pathogens, such as Gardnerella vaginalis, human papillomavirus, polyomavirus, etc.; in addition, as the infection proportion of drug-resistant bacteria in urinary tract infections gradually increases, existing drug-resistant gene detection technologies may not cover all important drug-resistant genes. Summary of the Invention

[0004] Aiming at the problems in the prior art, such as low detection sensitivity and specificity, long detection time, limited detection range, complex operation, high cost, and high false positive rate, and the problems of limited detection range of pathogenic microorganisms, low effective detection rate, and incomplete coverage of drug-resistant genes in the detection of existing urinary tract infections, especially symptomatic urinary tract infections with negative urine culture, the present invention aims to provide a primer combination, a kit and an application for detecting pathogenic microorganisms and drug-resistant genes in urinary tract infections.

[0005] To achieve the above object, the present invention is implemented by the following technical solutions: The present invention provides a primer combination for detecting pathogenic microorganisms and drug resistance genes in urinary tract infections. The pathogenic microorganisms include Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Enterobacter cloacae, Acinetobacter baumannii, Enterococcus faecium, Enterococcus faecalis, Pseudomonas maltophilia, Streptococcus agalactiae, Staphylococcus aureus, Serratia, Gardnerella vaginalis, Pseudomonas, Ureaplasma urealyticum, Candida, Human papillomavirus, Polyomavirus. The nucleotide sequences of the detection primers are shown in SEQ ID NO. 1 to SEQ ID NO. 38: The detection primers for at least 6 of the above pathogenic microorganisms are placed in one tube for sample detection. According to the different melting curve Tm values of the products, one tube is divided into three multiplex detection kit combinations, and each combination can at least achieve simultaneous detection of 6 urinary tract pathogenic microorganisms in a single tube; The drug resistance genes include mecA, QnrA, QnrB, OXA-10, blaCTX-M-1, BlaGES, BlaDHA, BlaCMY, BlaACT, cfxA, OXA-23, OXA-24, OXA-48, OXA-58, BlaIMP, BlaVIM, BlaKPC, BlaNDM, BlaSIM, oprD. The nucleotide sequences of the detection primers are shown in SEQ ID NO. 39 to SEQ ID NO. 78; The detection primers for at least 1 of the above drug resistance genes are placed in one tube for sample detection.

[0006] It also includes a detection primer for the internal reference gene GAPDH.

[0007] A kit for detecting pathogenic microorganisms and drug resistance genes in urinary tract infections, the kit contains the above primer combination for detecting pathogenic microorganisms and drug resistance genes in urinary tract infections.

[0008] The pathogenic microorganism detection primer combination is placed in one tube, and the drug resistance gene detection primer combination is placed in another tube.

[0009] The kit also includes a PCR reaction solution.

[0010] The kit also includes positive and negative control reagents.

[0011] The above primer combination for detecting pathogenic microorganisms and drug resistance genes in urinary tract infections, or the above kit for detecting pathogenic microorganisms and drug resistance genes in urinary tract infections is used in the in vitro non-disease diagnosis and treatment of urethral microorganism detection.

[0012] The present invention provides a method for detecting urethral microorganisms for non-disease diagnosis and treatment in vitro, including: extracting the DNA of the sample to be tested, performing a PCR amplification reaction using the primer combinations for detecting pathogenic microorganisms and drug resistance genes in urinary tract infections as described above, detecting the fluorescence signal, and determining whether these microorganisms or drug resistance genes are present in the sample.

[0013] The PCR amplification reaction system consists of a reaction buffer, deoxynucleoside triphosphate substrates (dNTPs), a heat-resistant DNA polymerase (Taq enzyme), oligonucleotide primers (forward primer, reverse primer), and a DNA template.

[0014] The PCR amplification reaction conditions are as follows: pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, annealing and extension at 60°C for 10 seconds, for a total of 40 cycles, with a stepwise temperature increase of 0.5°C to determine the melting curve.

[0015] The present invention uses the TB Green dye-embedded fluorescence method for Real Time PCR technology. High-specificity primers are designed based on the conserved sequences highly expressed in the detected microorganisms and drug resistance genes. While ensuring the detection specificity, it enables rapid PCR reactions and accurate quantification and detection of target genes within a wide dynamic quantification range, with good reaction repeatability.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The primer combinations for detecting pathogenic microorganisms and drug resistance genes in urinary tract infections provided by the present invention have technical characteristics such as multiplex detection ability, fluorescence labeling technology, and multi-detection in one tube. They can quickly and accurately detect various pathogenic microorganisms and drug resistance genes, improve the diagnostic efficiency, guide precise medication, and have strong adaptability. This is of great significance for the clinical diagnosis and treatment of urinary tract infections. The use of specific primers ensures the accuracy of detection, avoids false positives and false negatives, reduces the waiting time required by traditional culture methods. Based on the analysis of clinical case data, 3 kinds of microorganisms related to urinary tract infections are further screened out, solving the confusion of whether the newly detected microorganisms are pathogens, symbionts, or contaminants, assisting clinicians in making judgments, and enabling patients to obtain timely diagnosis and treatment.

[0017] The kit for detecting pathogenic microorganisms and drug resistance genes in urinary tract infections provided by the present invention has technical characteristics such as multiplex detection ability, high specificity and sensitivity, fluorescence labeling and quenching technology, and multi-detection in one tube. It can improve the diagnostic efficiency, guide precise medication, simplify the operation process and has strong adaptability; this is of great significance for the clinical diagnosis and treatment of urinary tract infections, and helps to improve the quality and efficiency of medical services; the kit supports multi-detection in one tube, simplifies the operation process and reduces the operation difficulty. At the same time, the primers in the kit have been optimized and verified to ensure the accuracy and reliability of the detection, reduce experimental errors and failure rates; it is applicable to patients of different ages, genders and disease conditions. As the pathogenic bacteria and drug resistance genes are constantly changing, the detection range of the kit can be further optimized and expanded to meet clinical needs.

[0018] The detection method provided by the present invention, through the comprehensive detection ability based on qPCR technology and the later screening and judgment system, this kit will significantly improve the sensitivity and specificity of the detection, reduce the false positive rate, and provide more powerful support for clinical treatment. Description of the Drawings

[0019] Figure 1 It is the detection reaction flow chart of the kit of the present invention; Figure 2 It is the relationship between the CT value detected by the kit of the present invention and the nucleic acid concentration of the sample; Figure 3 It is the culture and drug sensitivity results of CRAB in a clinical sample; among them, SCF is cefoxitin, TGC is tigecycline, TE is tetracycline, TZP is piperacillin / tazobactam, and AK is amikacin; Figure 4 It is the drug sensitivity detection result of this CRAB using the kit of the present invention; among them, (a) is the expression of DHA gene, the average CT value is 31.30, (b) is the expression of KPC gene, the average CT value is 33.09, (c) is Tm 81.5℃, (d) is Tm 84.5℃; Figure 5 The culture and drug sensitivity results of drug-resistant Proteus mirabilis in a clinical sample; among them, KZ is cefazolin, SCF is cefoxitin, TGC is tigecycline, TE is tetracycline, IPM is imipenem, MEM is meropenem, and CXM is cefuroxime; Figure 6Drug sensitivity test results of the Proteus mirabilis using the kit of the present invention; among them, (a) is the expression of the DHA gene, with an average CT value of 19.32, (b) is the low expression of the KPC gene, with an average CT value of 36.22, (c) is the low expression of the CTX-M gene, with an average CT value of 39.85, (d) Tm is 81.5 °C, (e) Tm is 84.5 °C, (f) Tm is 83.5 °C; Figure 7 Sensitivity test of the kit of the present invention; among them, (a) is the detection curve of the standard strain ATCC25922 simulating different sample concentrations in urine, positive control product and negative control product, (b) is the melting curve of the kit monitoring different sample concentrations in simulated urine, positive control product and negative control product; Figure 8 Sensitivity and stability of the kit of the present invention for simultaneously detecting multiple microorganisms in a single tube; among them, (a) is the amplification curve of simultaneously detecting 5 microorganisms in a single tube, (b) is the amplification curve of simultaneously detecting 6 microorganisms in a single tube, (c) is the melting curve of simultaneously detecting 5 microorganisms in a single tube, (d) is the melting curve of simultaneously detecting 6 microorganisms in a single tube; Figure 9 Univariate and multivariate logistic regression analysis of the present invention; among them, (a) is univariate analysis, (b) is multivariate analysis, and the results are expressed according to the odds ratio (OR) and 95% confidence interval (CI). Detailed implementation manners

[0020] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] The Proteus mirabilis (ATCC25933), Staphylococcus aureus (ATCC259231), Acinetobacter baumannii (ATCC19606), Escherichia coli (ATCC25922), Pseudomonas maltophilia (ATCC17666) and Gardnerella vaginalis (ATCC14018) adopted by the present invention are produced by the American Type Culture Collection (ATCC, www.atcc.org) and can be sold by agents such as Beijing Zhongyuan Heju Biotechnology Co., Ltd. and Shanghai Jitai Yikesai Biotechnology Co., Ltd.

[0022] The present invention will be further described in detail below in conjunction with the accompanying drawings, and the specific steps are as follows: Example 1 Design of primer sets for pathogenic microorganisms and drug-resistant genes The known sequences of Escherichia coli (EC), Klebsiella pneumoniae (KP), Streptococcus pneumoniae (LP), Pseudomonas aeruginosa (PA), Proteus mirabilis (PM), Enterobacter cloacae (EnC), Acinetobacter baumannii (AB), Enterococcus faecium (EFm), Enterococcus faecalis (EFs), Pseudomonas maltophilia (SM), Streptococcus agalactiae (SAg), Staphylococcus aureus (SAu), Serratia (SE), Gardnerella vaginalis (GV), Pseudomonas (PS), Ureaplasma urealyticum (UU), Candida (CA), Human papillomavirus (HPV), Polyomavirus (PV), quinolone resistance, cephalosporin resistance, carbapenem resistance, Methicillin-resistant Staphylococcus aureus (MRSA), and bacteria producing extended-spectrum β-lactamases (ESBLs) were compared and analyzed. The respective specific gene sequences were obtained from databases (such as GenBank, NCBI), and the sequences were aligned using bioinformatics tools (such as BLAST, ClustalW) to confirm the differential gene sequences. Detection primers were designed for the differential gene sequences, and the specific nucleotide sequences of the designed primers are shown as SEQ ID NO.1 to SEQ ID NO.78, and the amplified sequences are shown as SEQ ID NO.79 to SEQ ID NO.117.

[0023] Using the above-designed primers, a detection kit (PCR amplification system kit) was prepared, including a PCR reaction solution, a primer mixture, and a buffer, and the components are shown in Table 1 below: Table 1: Composition of the PCR amplification system kit

[0024] Example 2 The positive standard bacteria of 19 microorganisms were respectively cultured to an appropriate growth state, and the DNA of each strain was extracted as the template for PCR amplification; for the DNA template of each microorganism, 3 independent PCR amplification reactions were carried out using the kit, and the reaction operation process was as Figure 1 , and the melting temperature peak (Tm) and fluorescence signal intensity of each amplification were recorded. The microbial detection of the kit in Example 1 was repeated and verified with gradient dilution. The combination of this kit was used to detect the 19 kinds of microbial positive standard bacteria, and the detection was repeated 3 times, as shown in Table 2.

[0025] Table 2: Results of three repeated detections of each pathogenic microorganism and Tm values

[0026] As can be seen from the data in Table 2, the test results for 19 kinds of microbial positive reference strains showed that the results of the three repeated experiments were reliable, with good consistency and a coefficient of variation less than 3%, indicating a high repeatability of the experimental results. The peak melting temperature (Tm) was distributed between 74.5 and 85.5 °C, which was consistent with the expected results. The results of the serial dilution experiment showed that the kit could still detect the target microorganism under a relatively low concentration of DNA template, indicating its high sensitivity.

[0027] Group the primer combinations of the kit according to the Tm value, and at least 6 kinds of microorganisms can be detected simultaneously in a single tube. The specific combinations are shown in Table 3.

[0028] Table 3: Grouping of multiplex detection of the kit and types of microorganisms

[0029] Extract the genomic nucleic acid of EC, use a ultra-micro spectrophotometer (One drop OD1000+, Nanjing Wuyi Technology Co., Ltd.) to measure the concentration of the DNA extract, and determine that the OD260 / OD280 ratio ranges from 1.7 to 1.9. Use the detection kit of Example 1 to detect the DNA extract without dilution, diluted 2-fold, diluted 5-fold, diluted 10-fold, and diluted 50-fold respectively, record the CT value at each dilution, and estimate the relationship between the CT value and the corresponding nucleic acid concentration through the formula: nucleic acid (ng / uL) = -0.0305xCT value detected by the kit + 28.093. Draw a graph (attached Figure 2 ) and calculate the coefficient of determination R 2 .

[0030] As can be seen from the attached Figure 2 data, the coefficient of determination R 2 = 0.9608, indicating a good fit between the formula (or regression line) and the experimental data. Using this formula, the nucleic acid concentration in the sample can be accurately estimated. By substituting the measured CT value into the formula, the nucleic acid concentration of pathogenic microorganisms and drug resistance genes in the sample can be quickly estimated. This method is simple, fast, and suitable for rapid screening and preliminary analysis of a large number of samples.

[0031] Example 2 The purpose of this example is to verify the detection effect of the kit combination on drug-resistant bacteria infection through clinical sample detection, and compare it with the traditional urine bacterial culture and drug sensitivity test to evaluate the accuracy and reliability of the kit.

[0032] Two urine samples from patients with clinically drug-resistant bacterial urinary tract infections (UTIs) were selected and tested using this kit combination. Urine bacterial culture and drug sensitivity tests were performed. The specific method was as follows: Clinically collect urine samples from patients. Under sterile conditions, take 10 μL of urine specimens and inoculate them on blood plates and MacConkey agar plates respectively. The inoculation was carried out by the continuous streaking method. After inoculation, place them in an incubator at 37 °C for 24 h. Determine the drug sensitivity of the isolated bacterial colonies. The agar diffusion method sensitivity test (KB method) and the microbroth dilution method (MIC method) were used to determine the minimum inhibitory concentration (MIC) of bacteria against specific antibiotics. As a control method, the results were as follows:

Sample 1

[0033] Table 4: The drug sensitivity test results of this CRAB

[0034] Use the kit to perform drug sensitivity detection on CRAB, detect the expression of DHA and KPC genes respectively, record the changes in fluorescence signals during the amplification process, KB is the disk diffusion method, MIC is the minimum inhibitory concentration, S is sensitive, and R is resistant.

[0035] From the attached Figure 4 data, it can be seen that for the expression of the DHA gene: as the number of cycles increased, the fluorescence signal gradually increased, indicating that the DHA gene was successfully amplified. The average CT value was 31.30, which was relatively low, indicating a high expression level of the DHA gene in the sample; for the expression of the KPC gene: as the number of cycles increased, the fluorescence signal gradually increased, but the average CT value was 33.09, higher than the CT value of the DHA gene, indicating a relatively low expression level of the KPC gene in the sample; melting curve analysis: the Tm value of the DHA gene was 81.8 °C. At this temperature, the expression level of the DHA gene was high, indicating good stability. The Tm value of the KPC gene was 84.5 °C. Although it was also expressed, its stability was poor compared to the DHA gene.

[0036]

Sample 2

[0037] Table 5: Drug sensitivity test results of this Proteus mirabilis

[0038] KB is the disk diffusion method, MIC is the minimum inhibitory concentration, and R is resistance Example 3 The kit of the present invention was used to detect urine simulated samples to verify sensitivity and specificity.

[0039] Preparation of urine simulated samples: Take 10 mL of urine from healthy people, mix in bacterial solutions of EC (ATCC25922) with final concentrations of 50 cfu / mL, 500 cfu / mL, 5000 cfu / mL, and 50000 cfu / mL, and mix well to prepare simulated samples.

[0040] Take 1 mL of the simulated sample, add it to a 1.5 mL centrifuge tube, centrifuge at 8,000 rpm for 1 min at room temperature, discard the supernatant, and collect the precipitate. Add 180 µL of 20 mg / mL lysozyme solution (Thermo Scientific), incubate in a water bath at 65°C for 30 minutes, add 400 µL of lysis buffer Buffer Digestion (Sangon Biotech), and mix well by oscillation. Incubate in a water bath at 65°C for 30 minutes, add 200 µL of phosphate buffer (Buffer PB, Sangon Biotech), centrifuge at -20°C and 10,000 rpm for 5 min, take 500 µL of the supernatant, add 500 µL of isopropanol, centrifuge at 1,000 rpm for 5 min at room temperature, discard the supernatant, add 1 mL of 75% ethanol, invert and rinse for 1 min, centrifuge at 10,000 rpm for 2 min, discard the supernatant, and add 10 µL of Tris-EDTA (Sangon Biotech) Buffer to obtain the nucleic acid extract to be tested.

[0041] Add 1 µL of the sample nucleic acid to three wells, wells 1 to 4, of an eight-strip tube, and then add the EC detection solution (reaction solution, primers, buffer) of the kit described in Table 3 to the three wells to prepare a final 25 µL reaction system; add the corresponding EC detection solution of the kit described in Table 3 and a positive control product to well 5 as a positive control; add the corresponding EC detection solution of the kit described in Table 3 and a negative control product to well 6 as a negative control. Mix well and briefly centrifuge to collect the liquid to the bottom of the tube. Place the eight-strip into a CFX Connect PCR instrument (BioRad), select SYBR qPCR, and use Figure 1 the reaction procedure, and the results are as Figure 7 .

[0042] The kit of the present invention can effectively detect microbial infections with a final concentration of 50 cfu / mL.

[0043] Example 4 Verification of the sensitivity of the kit of the present invention for detecting microorganisms and drug-resistant genes. Further verification of the sensitivity of the kit of the present invention was carried out. Using the kit described in Example 1, the results were interpreted according to the threshold relationship shown in Example 2.

[0044] (1) Preparation of urine simulation samples Select the urine of healthy people as the basis, ensure that the urine does not contain the target pathogenic bacteria to avoid interfering with the experimental results. Mix 1 mL of the standard bacterial solutions of each pathogenic bacterium, namely Proteus mirabilis (ATCC25933), Staphylococcus aureus (ATCC25923), Acinetobacter baumannii (ATCC19606), Escherichia coli (ATCC25922), Gardnerella vaginalis (ATCC14018), and Pseudomonas maltophilia (ATCC17666), which have been cultured overnight, into 1 mL of urine from healthy people respectively. The selection of pathogenic bacteria should be based on the experimental purpose and the detection range of the kit to ensure that all pathogenic bacteria species that the kit can detect are covered.

[0045] Mix the urine mixed with pathogenic bacteria thoroughly to ensure the uniform distribution of pathogenic bacteria in the urine. The prepared simulation samples should be processed subsequently as soon as possible to avoid the reproduction or death of pathogenic bacteria affecting the experimental results.

[0046] (2) Nucleic acid extraction from samples Obtain a mixed solution of nucleic acid extracts using the nucleic acid extraction method in Example 2.

[0047] (3) Detection Take two 1-mL portions of the mixed solution of nucleic acid extracts and add them to wells 1 and 2 of an eight-well strip tube respectively. Use combination A shown in Table 5 and perform PCR reactions according to Figure 1 the procedure. Well 1 detects 5 microorganisms, namely Proteus mirabilis (ATCC25933), Staphylococcus aureus (ATCC25923), Acinetobacter baumannii (ATCC19606), Escherichia coli (ATCC25922), and Pseudomonas maltophilia (ATCC17666). Well 2 detects all 6 microorganisms, namely Proteus mirabilis (ATCC25933), Staphylococcus aureus (ATCC25923), Acinetobacter baumannii (ATCC19606), Escherichia coli (ATCC25922), Gardnerella vaginalis (ATCC14018), and Pseudomonas maltophilia (ATCC17666). The results are as shown in the appendix Figure 8 as follows.

[0048] The kit of the present invention can effectively detect multiple microbial infections simultaneously in a single tube. The detection results of 5 species are consistent with those of 6 species, and the detection sensitivity and stability are good.

[0049] Example 5 Verification of the stability of the kit of the present invention. All reagents used in the detection kit of the present invention are stored at -20 ± 3°C. Samples are taken after 0 month, 1 month, 3 months, and 6 months of storage. The positive samples are detected using the kit described in Example 1, and the amplification curves and fluorescence peaks are recorded. The results are shown in Table 6.

[0050] Table 6: Verification of the stability of this kit

[0051] It can be seen from the data in Table 6 that the kit of the present invention at different times can effectively detect the amplification curves and observe the target fluorescence peaks. It can be seen from the results that the detection kit of the present invention can be stored for at least 6 months in the environment of -20 ± 3°C, and its detection performance remains stable. This conclusion provides an important reference basis for the storage, transportation, and use of the kit, ensuring the reliability and accuracy of the kit in practical applications.

[0052] Example 6 In this example, the specificity of the kit of the present invention is analyzed and evaluated through the actual detection of urine samples from clinical UTI (urinary tract infection) patients.

[0053] Prepare 20 urine samples from clinical UTI patients. Each sample is separately subjected to urine culture, drug sensitivity test, next-generation sequencing, and combined detection using the kit of the present invention. The detection results of various detection methods are recorded and compared. The results are shown in Table 7.

[0054] Table 7: Detection results of 20 clinical urine samples

[0055] Urine culture takes at least 48 hours to get results. If a drug sensitivity test is done simultaneously with urine culture, it will take an additional 3 to 4 days to get results. At the same time, although next-generation sequencing can detect the most comprehensive types of microorganisms, it has high detection costs, takes a long time, and there are non-pathogenic microorganisms, host factors, laboratory contamination, environmental interference, etc. in the results that affect the detection. The combined detection of this kit generally only takes 4 - 6 hours, and the longest does not exceed 12 hours. Therefore, from the comparison of the detection results, the combined detection of this kit is more comprehensive than the combined detection of the clinical gold standard urine culture and drug sensitivity test, with a richer variety of microorganisms, and can effectively and highly specifically detect pathogenic microorganisms in urine, especially for the detection of multiple infections, which is more time-saving and labor-saving. Compared with next-generation sequencing, this kit is more cost-effective, has high detection efficiency and good specificity, and can exclude the influence of Prevotella, Lactobacillus, Bifidobacterium, Citrobacter, Bacteroides, etc. on the detection results while accurately detecting urinary tract pathogenic microorganisms.

[0056] See the appendix Figure 9 , and the results of univariate and multivariate logistic regression analysis revealed the association between urinary tract infection (UTI) and multiple pathogens. Compared with previous products, this kit newly added the detection of two new UTI-related pathogens, polyomavirus and Gardnerella vaginalis. In addition, it also covers urogenital mycoplasma and fungi, with a large detection range.

[0057] Analyze 65 urine samples with negative clinical bacterial culture, and use whole-exome sequencing (WGS) technology for detection and analysis. The established logistic regression model shows that, specifically see the appendix Figure 9 as shown.

[0058] From the appendix Figure 9 data, it can be seen that for univariate logistic regression analysis: Escherichia coli: the OR value is 11.688, 95% CI is 2.190 - 62.362, and the p value is 0.004, indicating that Escherichia coli is an independent risk factor for UTI; Gardnerella vaginalis: the OR value is 9.904, 95% CI is 2.180 - 45.005, and the p value is 0.003, also indicating that Gardnerella vaginalis is an independent risk factor for UTI; polyomavirus: the OR value is 5.205, 95% CI is 1.295 - 20.919, and the p value is 0.020, also showing a significant association with UTI; in addition to the known Escherichia coli being an important risk factor for UTI, Gardnerella vaginalis and polyomavirus also show a significant association with UTI.

[0059] Multivariate logistic regression analysis: Escherichia coli: In the multivariate analysis, the OR value of Escherichia coli remained significant, further confirming that it is an important risk factor for UTI. Gardnerella vaginalis also showed a significant association with UTI in the multivariate analysis, further supporting the conclusion that it is an independent risk factor for UTI; the OR value of polyomavirus in the multivariate analysis also reached a statistically significant level, indicating that it is also an important risk factor for UTI.

[0060] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution according to the technical idea proposed by the present invention fall within the protection scope of the claims of the present invention.

Claims

1. A primer combination for detecting pathogenic microorganisms and drug-resistant genes of urinary tract infections, characterized in that: The pathogenic microorganisms include Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Enterobacter cloacae, Acinetobacter baumannii, Enterococcus faecium, Enterococcus faecalis, Pseudomonas maltophilia, Streptococcus agalactiae, Staphylococcus aureus, Serratia, Gardnerella vaginalis, Pseudomonas, Ureaplasma urealyticum, Candida, human papillomavirus, polyomavirus, and the nucleotide sequences of the detection primers are shown in SEQ ID NO. 1 to SEQ ID NO. 38: The detection primers of at least 6 pathogenic microorganisms are placed in one tube for sample detection, and one tube is divided into three multiple detection kit combinations according to the different Tm values ​​of the product melting curves, and each combination can realize at least 6 urinary tract pathogenic microorganisms to be detected simultaneously in a single tube; The drug-resistant genes include mecA, QnrA, QnrB, OXA-10, blaCTX-M-1, BlaGES, BlaDHA, BlaCMY, BlaACT, cfxA, OXA-23, OXA-24, OXA-48, OXA-58, BlaIMP, BlaVIM, BlaKPC, BlaNDM, BlaSIM, and oprD, and the nucleotide sequences of the detection primers are shown in SEQ ID NO. 39 to SEQ ID NO. 78; The detection primers of at least one drug-resistant gene are placed in a tube for sample detection.

2. The primer combination for detecting pathogenic microorganisms and drug-resistant genes of urinary tract infection according to claim 1, characterized in that: Also included are detection primers for the internal reference gene GAPDH.

3. A kit for detecting pathogenic microorganisms and drug-resistant genes of urinary tract infections, characterized in that: The kit contains the primer combination for detecting urinary tract infection pathogenic microorganisms and drug-resistant genes as described in claim 1 or 2.

4. The kit for detecting pathogenic microorganisms and drug-resistant genes of urinary tract infection according to claim 3, characterized in that: The pathogenic microorganism detection primer combination is placed in one tube, and the drug resistance gene detection primer combination is placed in another tube.

5. The kit for detecting pathogenic microorganisms and drug-resistant genes of urinary tract infection according to claim 3, characterized in that: The kit also includes a PCR reaction solution.

6. The kit for detecting pathogenic microorganisms and drug-resistant genes of urinary tract infection according to claim 3, characterized in that: The kit also includes positive quality control and negative quality control reagents.

7. Use of the primer combination for detecting pathogenic microorganisms and drug-resistant genes of urinary tract infection according to any one of claims 1 to 2, or the kit for detecting pathogenic microorganisms and drug-resistant genes of urinary tract infection according to any one of claims 3 to 6 in in vitro non-disease diagnosis and treatment of urinary tract microorganism detection.

8. A method for in vitro non-disease diagnosis and treatment of urinary tract microorganisms, characterized in that: include: Extract the DNA of the sample to be tested, perform PCR amplification reaction using the primer combination for detecting urinary tract infection pathogenic microorganisms and drug-resistant genes as described in any one of claims 1 to 2, detect the fluorescent signal, and determine whether these microorganisms or drug-resistant genes are present in the sample.

9. The method for in vitro non-disease diagnosis and treatment of urinary tract microorganisms according to claim 8, characterized in that: The PCR amplification reaction system consists of a reaction buffer, a deoxynucleoside triphosphate substrate, a heat-resistant DNA polymerase, an oligonucleotide primer and a DNA template.

10. The method for in vitro non-disease diagnosis and treatment of urinary tract microorganisms according to claim 8, characterized in that: The PCR amplification reaction conditions are: 95°C for 30 seconds pre-denaturation, 95°C for 5 seconds denaturation, 60°C for 10 seconds annealing and extension, a total of 40 cycles, 0.5°C step temperature increase, and determination of the melting curve.

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