Composition and kit for simultaneously detecting urinary tract infection pathogenic bacteria and drug-resistant genes and application of composition and kit

Abstract

The invention belongs to the field of molecular biology detection, and particularly relates to a composition and a method for detecting and distinguishing proteus mirabilis, klebsiella pneumoniae, escherichia coli and a fosfomycin drug-resistant gene GlpT gene, a fluoroquinolone drug-resistant gene GyrA gene and a fluoroquinolone drug-resistant gene GyrB gene, and application of the composition and the method. The joint detection composition provided by the invention can realize rapid detection of three pathogen target genes and three drug-resistant gene segments on pathogens in one tube of reaction liquid by utilizing a PCR detection technology, and has the characteristics of short time, high sensitivity, good specificity and the like. According to the invention, pathogenic bacteria can be accurately detected, and the mixed infection condition of bacteria can be analyzed, so that the effect of accurate medication is achieved, the use of antibiotics is reduced, and the cost is saved.

Description

Technical Field

[0001] The present invention relates to the field of molecular biological detection, and specifically, to the detection of urinary tract infection pathogens and drug-resistant genes; more specifically, to the detection of Proteus mirabilis, Klebsiella pneumoniae, Escherichia coli and fosfomycin resistance gene GlpT gene, fluoroquinolone drug resistance gene GyrA gene, and fluoroquinolone drug resistance gene GyrB gene. Background Art

[0002] Proteus mirabilis is a Gram-negative bacterium belonging to the Enterobacteriaceae family. It is widely distributed in nature (such as soil, water, and corrupted organic matter) and also exists in the intestines of humans and animals. It is a conditional pathogen and an important pathogen in urinary tract infection (UTI). Proteus mirabilis mainly causes urinary tract infection through retrograde infection of the urethra and is one of the common pathogens of hospital-acquired urinary tract infection. The isolation rate of Proteus mirabilis is relatively high in community-acquired urinary tract infections. Especially in specific populations, such as young women, the elderly, and people with weakened immunity, uncomplicated urinary tract infections caused by Proteus mirabilis are more common.

[0003] Klebsiella pneumoniae is a Gram-negative bacillus and the most important type of bacteria in the genus Klebsiella of the family Enterobacteriaceae (commonly known as pneumonia bacillus). Klebsiella can be isolated from the normal flora of the respiratory tract and intestines of healthy people, and from natural water and grains. Klebsiella pneumoniae is one of the important pathogens that cause uncomplicated urinary tract infections (UTIs). Although its incidence in urinary tract infections is relatively lower than that of common pathogens such as Escherichia coli, it is still relatively common in specific populations. For example, the incidence of uncomplicated urinary tract infections caused by Klebsiella pneumoniae is increased in patients with long-term indwelling catheters, low immune function, and underlying diseases such as diabetes.

[0004] Escherichia coli is a Gram-negative bacillus, also known as Escherichia coli, and is a species of the genus Escherichia in the family Enterobacteriaceae. Escherichia coli is the most common pathogen causing urinary tract infection (UTI). In community-acquired urinary tract infections, the isolation rate of Escherichia coli can be as high as 70% to 90%. Escherichia coli plays an important role in both simple and complicated urinary tract infections. It can cause urinary tract infections in people of all ages, but is more common in women, the elderly, and people with weakened immune systems.

[0005] Fosfomycin is a broad-spectrum antibiotic that is effective against Gram-positive and Gram-negative bacteria, has high antibacterial activity against Escherichia coli, and also has certain antibacterial effects against Proteus and Klebsiella pneumoniae. Especially in the treatment of urinary tract infections, it has a good effect on infections caused by Escherichia coli because it can maintain a high drug concentration in urine, thereby effectively inhibiting bacteria. The glycerol-3-phosphate transporter encoded by the GlpT gene plays a key role in the sensitivity of these three Enterobacteriaceae to fosfomycin. Detecting the status of the GlpT gene in bacteria can be used as a rapid and effective method to determine the resistance of bacteria to fosfomycin.

[0006] Since their introduction in the 1960s, fluoroquinolone antibiotics have gone through several stages of development. At present, they have a wider antibacterial spectrum and have strong antibacterial effects on both Gram-positive and Gram-negative bacteria. They have strong antibacterial effects on common Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa. Fluoroquinolone antibiotics mainly inhibit bacterial DNA gyrase (topoisomerase II) and topoisomerase IV, interfering with the replication, transcription, and repair process of bacterial DNA, thereby exerting a bactericidal effect. In the process of bacterial resistance to fluoroquinolones, GyrA and GyrB genes often mutate. Among them, mutations in the GyrA gene are more common, mainly occurring in the quinolone resistance determining region (QRDR). For example, in bacteria such as Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis, common mutation sites include Ser83→Leu, Asp87→Asn / Gly / Tyr, etc. Mutations in the GyrB gene are relatively rare, but may also occur in some specific regions, affecting the function of DNA gyrase and the binding of fluoroquinolones.

[0007] It is worth noting that three Enterobacteriaceae, Proteus mirabilis, Klebsiella pneumoniae, and Escherichia coli, are closely associated with resistance to fosfomycin by the GlpT gene and resistance to fluoroquinolones by the GyrA and GyrB genes. Therefore, it is very important to confirm the pathogen, the co-resistant fosfomycin genotype (GlpT), and the co-resistant fluoroquinolone genotype (GyrA, GyrB) before treatment. This ensures effective treatment and reduces the development of fosfomycin and fluoroquinolone resistance.

[0008] Bacterial culture is the gold standard for the diagnosis of pathogens, but the isolation and culture of pathogens is time-consuming and cumbersome. Compared with other detection methods, real-time fluorescence quantitative PCR technology is more suitable for early detection, with high sensitivity and specificity, and can accurately and quickly detect Proteus mirabilis, Klebsiella pneumoniae, Escherichia coli, resistant fosfomycin genotypes (GlpT) and resistant fluoroquinolone genotypes (GyrA, GyrB). Summary of the invention

[0009] In view of this, in a first aspect, the present invention provides a composition for simultaneously detecting urinary tract infection pathogens and drug resistance genes, the composition comprising three of the following primers and probes for detecting urinary tract infection pathogens:

[0010] The upstream and downstream primers and probes of Klebsiella pneumoniae as shown in SEQ ID NOs: 1 to 3;

[0011] Upstream and downstream primers and probes of Proteus mirabilis as shown in SEQ ID NOs: 4 to 6;

[0012] The upstream and downstream primers and probes of Escherichia coli as shown in SEQ ID NOs: 7 to 9;

[0013] The composition also includes at least one of primers and probes for detecting the following drug resistance genes:

[0014] Upstream and downstream primers and probes of the fosfomycin resistance gene GlpT as shown in SEQ ID NOs: 10 to 12;

[0015] Upstream and downstream primers and probes of the fluoroquinolone resistance gene GyrA as shown in SEQ ID NOs: 13 to 15;

[0016] The upstream and downstream primers and probes of the fluoroquinolone resistance gene GyrB as shown in SEQ ID NOs: 16-18.

[0017] Further, a composition for simultaneously detecting urinary tract infection pathogens and drug resistance genes, the composition comprising three of the following primers and probes for detecting urinary tract infection pathogens:

[0018] The upstream and downstream primers and probes of Klebsiella pneumoniae as shown in SEQ ID NOs: 1 to 3;

[0019] Upstream and downstream primers and probes of Proteus mirabilis as shown in SEQ ID NOs: 4 to 6;

[0020] The upstream and downstream primers and probes of Escherichia coli as shown in SEQ ID NOs: 7 to 9;

[0021] The composition also includes at least two of the primers and probes for detecting the following drug resistance genes:

[0022] Upstream and downstream primers and probes of the fosfomycin resistance gene GlpT as shown in SEQ ID NOs: 10 to 12;

[0023] Upstream and downstream primers and probes of the fluoroquinolone resistance gene GyrA as shown in SEQ ID NOs: 13 to 15;

[0024] The upstream and downstream primers and probes of the fluoroquinolone resistance gene GyrB as shown in SEQ ID NOs: 16-18.

[0025] Further, a composition for simultaneously detecting urinary tract infection pathogens and drug resistance genes, the composition comprising three of the following primers and probes for detecting urinary tract infection pathogens:

[0026] The upstream and downstream primers and probes of Klebsiella pneumoniae as shown in SEQ ID NOs: 1 to 3;

[0027] Upstream and downstream primers and probes of Proteus mirabilis as shown in SEQ ID NOs: 4 to 6;

[0028] The upstream and downstream primers and probes of Escherichia coli as shown in SEQ ID NOs: 7 to 9;

[0029] The composition also includes three of the primers and probes for detecting the following drug resistance genes:

[0030] Upstream and downstream primers and probes of the fosfomycin resistance gene GlpT as shown in SEQ ID NOs: 10 to 12;

[0031] Upstream and downstream primers and probes of the fluoroquinolone resistance gene GyrA as shown in SEQ ID NOs: 13 to 15;

[0032] The upstream and downstream primers and probes of the fluoroquinolone resistance gene GyrB as shown in SEQ ID NOs: 16-18.

[0033] The combined detection composition provided by the present invention mainly utilizes PCR detection technology, can realize rapid detection of three pathogen target genes and three drug-resistant gene fragments in one tube of reaction liquid, is suitable for early detection, has high sensitivity and specificity, and can accurately and rapidly detect Proteus mirabilis, Klebsiella pneumoniae, Escherichia coli, fosfomycin resistance gene GlpT, fluoroquinolone resistance gene GyrA and fluoroquinolone resistance gene GyrB.

[0034] Furthermore, in the composition, the fluorescent groups of the probes are different from each other and do not interfere with each other.

[0035] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, FAM, HEX, ROX and CY5 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected, so they will not interfere with each other.

[0036] In the present invention, the fluorescent reporter group may be selected from FAM, HEX, ROX, VIC, CY5, ATTO 425, Quasar705, 5-TAMRA, TET, CY3 and JOE, but is not limited thereto.

[0037] In a specific embodiment, the fluorescent reporter group of the probe for Klebsiella pneumoniae as shown in SEQ ID NO: 3 is FAM; the fluorescent reporter group of the probe for Proteus mirabilis as shown in SEQ ID NO: 6 is HXE; the fluorescent reporter group of the probe for Escherichia coli as shown in SEQ ID NO: 9 is ROX; the fluorescent reporter group of the probe for the fosfomycin resistance gene GlpT as shown in SEQ ID NO: 12 is CY5; the fluorescent reporter group of the probe for the fluoroquinolone resistance gene GyrA as shown in SEQ ID NO: 15 is ATTO 425; the fluorescent reporter group of the probe for the fluoroquinolone resistance gene GyrB as shown in SEQ ID NO: 18 is Quasar 705.

[0038] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1, BHQ2, or MGB.

[0039] In a specific embodiment, the 3' end of the probe is BHQ1.

[0040] In a specific embodiment, the 3' end of the probe is BHQ2.

[0041] Further, in some embodiments, the composition of the present invention may include one or more pairs of the above primer and probe pairs. In the present invention, a "pair" refers to mutually matching upstream and downstream primers and probes for detecting a target.

[0042] Furthermore, the amount of the primer in the composition is 0.05 μM-5 μM; the amount of the probe in the composition is 0.05 μM-5 μM.

[0043] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0044] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0045] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0046] In a second aspect, the present invention provides the use of the above-mentioned composition of the present invention in the preparation of a kit for simultaneously detecting urinary tract infection pathogens and drug-resistant genes, wherein the urinary tract infection pathogens are Klebsiella pneumoniae, Proteus mirabilis, and Escherichia coli; and the drug-resistant genes are fosfomycin resistance gene GlpT, fluoroquinolone resistance gene GyrA, and fluoroquinolone resistance gene GyrB.

[0047] Furthermore, the kit is used for fluorescent PCR detection.

[0048] In a third aspect, the present invention provides a kit for simultaneously detecting urinary tract infection pathogens and drug-resistant genes, wherein the kit comprises the composition of the present invention as described above.

[0049] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0050] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTPs (U), UNG enzyme (uracil glycosylase), DNA polymerase, PCR buffer and Mg 2+ At least one of .

[0051] In a specific embodiment, the DNA polymerase is H-Taq enzyme.

[0052] Common PCR buffers are composed of Tris-HCl, MgCl2, KCl, Triton X-100, etc. The total volume in a single PCR reaction tube is generally 20 μL to 100 μL.

[0053] In a specific embodiment, the kit of the present invention is compatible with a digital PCR amplification system, that is, it can be directly used for amplification on a digital PCR instrument.

[0054] In a fourth aspect, the present invention provides a use of a composition for preparing a detection kit for simultaneously detecting urinary tract infection pathogens and drug resistance genes, wherein the detection comprises the following steps:

[0055] S1. Extract or release nucleic acid from the sample to be tested;

[0056] S2. Performing fluorescent quantitative PCR analysis on the nucleic acid obtained in step S1 using the composition of the present invention or the kit of the present invention;

[0057] S3. Obtain and analyze the results.

[0058] In a specific embodiment, a composition is provided for preparing a detection kit for simultaneously detecting urinary tract infection pathogens and drug resistance genes, wherein the detection comprises the following steps:

[0059] S1. Extract or release nucleic acid from the sample to be tested;

[0060] S2. Performing fluorescence quantitative PCR analysis on the nucleic acid obtained in step S1 using the composition of the present invention or the kit of the present invention; further, the reaction conditions of the fluorescence quantitative PCR include: UNG enzyme reaction at 50°C, 2 minutes, 1 cycle; H-Taq enzyme activation at 94°C, 3 minutes, 1 cycle; denaturation at 94°C, 15 seconds, annealing, extension, fluorescence collection at 60°C, 15 seconds, 45 cycles.

[0061] S3. Obtain and analyze the results. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is a graph showing the test results of the composition of the present invention;

[0063] Figure 2 The sensitivity test results of the composition of the present invention are as follows;

[0064] Figure 3 is the specific detection result of the composition of the present invention;

[0065] Figure 4 The test results of the five-combination test of the composition of the present invention;

[0066] Figure 5 The test results of the quadruple test of the composition of the present invention;

[0067] Figure 6 The result of single detection of Klebsiella pneumoniae by the detection system after replacing the Klebsiella pneumoniae primers and probes in Comparative Example 1 of the present invention;

[0068] Figure 7 This is the result of the joint detection of the detection system after replacing the Klebsiella pneumoniae primers and probes in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0069] To make the present invention easier to understand, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by professional and technical personnel in the field; unless otherwise specified, the instruments and reagents involved in this article are conventional instruments and reagents in the field.

[0070] Example 1: Primers and probes used in the present invention

[0071] Table 1:

[0072]

[0073] Among them, the fluorescent reporter group of the probe for Klebsiella pneumoniae is FAM; the fluorescent reporter group of the probe for Proteus mirabilis is HXE; the fluorescent reporter group of the probe for Escherichia coli is ROX; the fluorescent reporter group of the probe for the fosfomycin resistance gene GlpT is CY5; the fluorescent reporter group of the probe for the fluoroquinolone resistance gene GyrA is ATTO425; and the fluorescent reporter group of the probe for the fluoroquinolone resistance gene GyrB is Quasar705.

[0074] Example 2: Method for Simultaneous Detection of Urinary Tract Infection Pathogens and Drug Resistance Genes

[0075] (1) Reagent preparation:

[0076] Take the corresponding amount of PCR buffer and enzyme mixture (PCR buffer 38 μL / person + enzyme mixture 2 μL / person) in proportion and mix them thoroughly to form a PCR mixture. The negative control is sterile saline; the mixed plasmid containing the corresponding specific gene target sequence is the positive control.

[0077] PCR buffer is composed of a buffer system including Tris-HCl, MgCl2, KCl, and TritonX-100.

[0078] The enzyme mixture consists of UNG enzyme and H-Taq enzyme. H-Taq enzyme (5U / μL) and UNG enzyme (2U / μL) are mixed in a certain ratio (1.9μL H-Taq enzyme and 0.1μL UNG enzyme per person). The enzyme mixture is used in an amount of 2μL per person.

[0079] (2) Sample processing and sample addition:

[0080] The sample to be tested in the embodiment of the present invention is urine. 300uL of the sample to be tested, the negative control, and the positive control are respectively taken into 1.5mL centrifuge tubes, and nucleic acid extraction is performed using commercially available nucleic acid extraction or purification reagents from Shengxiang Biotechnology Co., Ltd. according to the instructions.

[0081] 10uL of the above-treated samples, negative controls and positive controls were pipetted into reaction tubes respectively, and 40uL of PCR mixed solution was added to each reaction tube and the tube cap was covered.

[0082] The reaction systems of the above-mentioned original reaction tubes are shown in Table 2 below:

[0083] Table 2:

[0084] Components Volume / concentration in each reaction PCR Buffer 34.3 dNTPs(U)(100mM) 0.5 <![CDATA[1mol / LMgcl2]]> 0.2 Primer SEQ ID NO: 1 (50 pmol / μL) 0.2 Primer SEQ ID NO: 2 (50 pmol / μL) 0.2 Primer SEQ ID NO: 4 (50 pmol / μL) 0.1 Primer SEQ ID NO: 5 (50 pmol / μL) 0.2 Primer SEQ ID NO:7 (50 pmol / μL) 0.2 Primer SEQ ID NO: 8 (50 pmol / μL) 0.1 Primer SEQ ID NO: 10 (50 pmol / μL) 0.2 Primer SEQ ID NO: 11 (50 pmol / μL) 0.2 Primer SEQ ID NO: 13 (50 pmol / μL) 0.1 Primer SEQ ID NO: 14 (50 pmol / μL) 0.2 Primer SEQ ID NO: 16 (50 pmol / μL) 0.2 Primer SEQ ID NO: 17 (50 pmol / μL) 0.1 Probe SEQ ID NO: 3 (50 pmol / μL) 0.2 Probe SEQ ID NO: 6 (50 pmol / μL) 0.2 Probe SEQ ID NO: 9 (50 pmol / μL) 0.1 Probe SEQ ID NO: 12 (50 pmol / μL) 0.2 Probe SEQ ID NO: 15 (50 pmol / μL) 0.2 Probe SEQ ID NO: 18 (50 pmol / μL) 0.1

[0085] (3) PCR amplification:

[0086] On the fully automatic medical PCR analysis system, PCR amplification is performed according to a certain temperature and time setting program. Among them, the reaction parameters of PCR amplification are shown in Table 3 below:

[0087] Table 3:

[0088]

[0089] (4) Result judgment:

[0090] If the FAM, HEX, CY5, ROX, ATTO425, Quasar705 channels have obvious S-shaped amplification curves and the CT value is ≤38, it is judged as positive; if the FAM, HEX, CY5, ROX, ATTO425, Quasar705 channels have no amplification curve (NoCT) or the CT value is greater than 38, it is judged as negative. The details are shown in Table 4:

[0091] Table 4:

[0092]

[0093] Example 3: Test results of the test samples of the composition of the present invention

[0094] The composition was verified, and the primers and probes shown in Example 1 were used according to the method of Example 2 to perform joint detection and differentiation on pathogens that were positive for various pathogens (genes of Klebsiella pneumoniae, Proteus mirabilis, and Escherichia coli) and resistant (fosfomycin resistance gene GlpT, fluoroquinolone resistance gene GyrA, and fluoroquinolone resistance gene GyrB). The test results were as follows: Figure 1 As shown. Figure 1 It can be seen that the composition of the present application can simultaneously detect urinary tract infection pathogens and drug-resistant genes.

[0095] Example 4: Sensitivity of the composition of the present invention

[0096] The positive samples were diluted with negative samples to a concentration of 200 copies / mL to verify the sensitivity of the composition and kit of the present invention. Figure 2 As shown in Table 5 below, the test results show that the composition can still accurately detect each channel of samples below 200 copies / mL. The results show that the method of the present invention has high sensitivity and the detection concentration can reach 200 copies / mL.

[0097] Table 5:

[0098]

[0099] Example 5. Specificity of the composition of the present invention

[0100] The primers and probes shown in Example 1 were used in accordance with the method described in Example 2 to perform multiplex PCR detection on a fluorescent quantitative PCR instrument for other urinary tract infection pathogens (Staphylococcus aureus, Neisseria gonorrhoeae, Mycoplasma, Chlamydia, Citrobacter rodentium, Enterococcus faecalis and Pseudomonas aeruginosa) that have homology in nucleic acid sequences and are likely to cause the same or similar clinical symptoms. The results are shown in FIG. Figure 3 As shown, from Figure 3 It can be seen that the detection of each target point is negative, which proves that the composition of the present invention has good specificity.

[0101] Example 6: Anti-interference performance of the composition of the present invention

[0102] The kit of Example 2 was analyzed for interferences by conventional methods. The experimental results showed that potential PCR inhibitors / interfering substances such as dexamethasone (50 μg / mL), gentamicin (50 μg / mL), vancomycin (50 μg / mL), cefmenoxime hydrochloride (50 μg / mL), ciprofloxacin (50 μg / mL), cotrimoxazole (50 μg / mL), clindamycin (50 μg / mL), heme (10 μg / mL), hemoglobin (10%), etc. at certain concentrations had no significant effect on the kit. The amplification results of the PCR reaction solution in the presence of infectious substances are specifically shown in Table 6 below:

[0103] Table 6:

[0104]

[0105]

[0106] Example 7, five-combination test of the composition of the present invention

[0107] The primers and probes shown in Example 1 were used to perform a five-joint test and differentiate Klebsiella pneumoniae, Proteus mirabilis, Escherichia coli, fosfomycin resistance gene GlpT, and fluoroquinolone resistance gene GyrA according to the method of Example 2. The test results are as follows: Figure 4 As shown. Figure 4 It can be seen that the composition of the present application can simultaneously perform five-in-one tests on urinary tract infection pathogens and drug-resistant genes.

[0108] Example 8: Quadruple test of the composition of the present invention

[0109] The primers and probes shown in Example 1 were used to perform quadruple detection and differentiation on Klebsiella pneumoniae, Proteus mirabilis, Escherichia coli, and the fosfomycin resistance gene GlpT according to the method of Example 2. The detection results are as follows: Figure 5 As shown. Figure 5It can be seen that the composition of the present application can simultaneously perform quadruple detection on urinary tract infection pathogens and drug resistance genes.

[0110] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0111] Due to the principle of complementary base pairing, dimers will form between primers and (or) probes, but this probability is very small and can be ruled out at the beginning of the design. However, when multiple pathogens are detected together, there are many primers and probes, and dimers are likely to occur between primers and primers, probes and probes, or primers and probes. In order to ensure the conservatism of the design (conservatism is crucial to the accuracy of the detection), and to consider the mutual interference between different primers and probes, the primers and probes need to be carefully designed.

[0112] Therefore, the inventors also designed some other primers and probes, such as another pair of Klebsiella pneumoniae primers and probes (upper and downstream primers and probes are shown in SEQ ID NOs: 19 to 21, respectively), which were replaced with the Klebsiella pneumoniae primers and probes in the kit of the present invention. The replaced detection system was used to detect Klebsiella pneumoniae alone, and the detection results were as follows: Figure 6 The replaced detection system was used to perform joint detection on the specific genes of Proteus mirabilis, Klebsiella pneumoniae, Escherichia coli, GlpT, GyrA, and GyrB, and the results are shown in Figure 7 As shown by Figure 6-7 As shown, although the detection performance of the detection system of this comparative example and the results of single detection of Klebsiella pneumoniae are similar to that of the present application, when they are subjected to joint detection, the detection performance of Klebsiella pneumoniae is reduced, the CT value of (Proteus mirabilis, Klebsiella pneumoniae, Escherichia coli, GlpT, GyrA, GyrB specific gene) lags behind, and the fluorescence increment of (Proteus mirabilis, Klebsiella pneumoniae, Escherichia coli, GlpT, GyrA, GyrB specific gene) decreases.

[0113] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A composition for simultaneously detecting urinary tract infection pathogens and drug resistance genes, characterized in that: The composition includes three of the following primers and probes for detecting urinary tract infection pathogens: The upstream and downstream primers and probes of Klebsiella pneumoniae as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes of Proteus mirabilis as shown in SEQ ID NOs: 4 to 6; The upstream and downstream primers and probes of Escherichia coli as shown in SEQ ID NOs: 7 to 9; The composition also includes at least one of primers and probes for detecting the following drug resistance genes: Upstream and downstream primers and probes of the fosfomycin resistance gene GlpT as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes of the fluoroquinolone resistance gene GyrA as shown in SEQ ID NOs: 13 to 15; The upstream and downstream primers and probes of the fluoroquinolone resistance gene GyrB as shown in SEQ ID NOs: 16-18.

2. The composition according to claim 1, characterized in that In the composition, the fluorescent groups of the probes are different from each other and do not interfere with each other.

3. The composition according to claim 2, characterized in that The fluorescent reporter group of the probe for Klebsiella pneumoniae as shown in SEQ ID NO: 3 is FAM; The fluorescent reporter group of the probe of Proteus mirabilis as shown in SEQ ID NO: 6 is HXE; The fluorescent reporter group of the probe for E. coli as shown in SEQ ID NO: 9 is ROX; The fluorescent reporter group of the probe of the fosfomycin resistance gene GlpT as shown in SEQ ID NO: 12 is CY5; The fluorescent reporter group of the probe of the fluoroquinolone resistance gene GyrA as shown in SEQ ID NO: 15 is ATTO425; As shown in SEQ ID NO: 18, the probe fluorescent reporter group of the fluoroquinolone resistance gene GyrB is Quasar705.

4. The composition according to claim 1, characterized in that The composition is present in a mixed form.

5. Use of the composition according to any one of claims 1 to 4 in the preparation of a kit for simultaneously detecting urinary tract infection pathogens and drug-resistant genes.

6. The use according to claim 5, wherein the kit is a fluorescent PCR detection kit.

7. The use according to claim 5, wherein the urinary tract infection pathogens are Klebsiella pneumoniae, Proteus mirabilis, and Escherichia coli; and the drug resistance genes are fosfomycin resistance gene GlpT, fluoroquinolone resistance gene GyrA, and fluoroquinolone resistance gene GyrB.

8. A kit for simultaneously detecting urinary tract infection pathogens and drug resistance genes, characterized in that: The kit comprises the composition according to any one of claims 1 to 4.

9. The kit according to claim 8, characterized in that The kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTPs, UNG enzyme, DNA polymerase, PCR buffer and Mg 2+ At least one of .

10. Use of a composition for preparing a detection kit for simultaneously detecting urinary tract infection pathogens and drug resistance genes, characterized in that: The detection comprises the following steps: S1. Extract or release nucleic acid from the sample to be tested; S2. Performing fluorescent quantitative PCR analysis on the nucleic acid obtained in step S1 using the composition according to any one of claims 1 to 4 or the kit according to any one of claims 8 to 9; S3. Obtain and analyze the results.

Images