Composition for detecting urinary tract infection related pathogens and drug resistance thereof, kit and application

Abstract

The invention belongs to the field of molecular biological detection, particularly relates to detection of urinary tract infection related pathogens and drug resistance thereof, and provides a combined detection composition which mainly utilizes a multiple fluorescent PCR analysis method to detect different targets by detecting the targets on the different pathogens so as to detect the drug resistance of the urinary tract infection related pathogens and the drug resistance of the urinary tract infection related pathogens. Therefore, detection and distinguishing of citrobacter freundii, proteusbacillus vulgaris, providencia, AmpC genes, TEM genes, NDM-1 genes and NDM-2 genes are realized in a single-tube reaction system at the same time, so that a targeted strategy is provided for subsequent treatment. The composition disclosed by the invention is higher in detection sensitivity (up to 200 copies / mL), good in specificity and more accurate in detection, so that clinicians can specifically provide strategies for prevention and treatment of infection caused by the pathogens.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to the detection of urinary tract infection-related pathogens and their drug resistance, and more specifically, to the detection of three pathogens, namely, Citrobacter freundii, Proteus vulgaris, and Providencia, and drug resistance genes AmpC gene, TEM gene, and NDM gene. Background Art

[0002] Citrobacter freundii is a Gram-negative aerobic or facultative anaerobic bacterium. Citrobacter freundii can cause food poisoning and diarrhea in humans, and can also cause clinical symptoms such as urethritis, wound infection, necrotizing abscess, peritonitis, pancreatitis, necrotizing fasciitis, osteomyelitis, pneumonia, pharyngitis, nephritis, bacteremia, infantile meningitis, brain abscess, etc. The urethra and intestines are the most susceptible parts to infection. The drug resistance rate of Citrobacter freundii is gradually increasing, and the number of drug-resistant strains producing carbapenemase, extended-spectrum β-lactamase (ESBLs) and cephalosporinase (AmpC enzyme) is increasing. The difficulty of clinical treatment and the incidence of poor prognosis are also gradually increasing.

[0003] Proteus vulgaris is a Gram-negative facultative anaerobic bacterium, a conditional pathogen. The probability of causing urinary tract infection is second only to Escherichia coli. Nosocomial infections are common, such as indwelling catheters, urinary tract obstruction, intestinal bacterial migration, etc., which cause many human infections. It can cause bacteremia and deep infections, especially in the ear, mastoid sinus, peritoneal cavity, and urinary tract of patients with chronic urinary tract infection or kidney or bladder stones; Proteus vulgaris can produce urease, hydrolyze urea, lead to alkaline urine, and thus form struvite (magnesium ammonium phosphate stones). It is easy to develop drug resistance, but is usually sensitive to fluoroquinolones, carbapenems, piperacillin-tazobactam, third-generation cephalosporins, and cefixime.

[0004] Providencia Ewing is a Gram-negative facultative anaerobic bacterium that is widely distributed in the environment. It is a conditional pathogen that often causes diarrhea and extraintestinal infections, such as urinary tract infections, meningitis, and sepsis. Urinary tract infections with Providencia Ewing can cause symptoms including urethral burning, frequent urination, urgency, dysuria, and urethral discharge. In severe cases, it can lead to complications such as renal scarring and chronic renal failure. In recent years, the detection rate of multidrug-resistant Providencia bacteria has continued to increase, and infections caused by carbapenem-resistant Providencia have continued to appear, making it difficult to choose therapeutic drugs.

[0005] The above pathogens are all prone to antibiotic resistance. Therefore, this field needs to detect the above pathogens while also clarifying their resistance, especially the resistance to third-generation cephalosporins and carbapenems.

[0006] Therefore, there is a need in the art for a product that can simply and quickly detect the above-mentioned pathogens and the corresponding drug-resistant genes, and has good sensitivity and specificity, and can detect and distinguish the pathogens that cause infection and their drug resistance, so as to provide targeted strategies for the prevention and treatment of infections caused by the above-mentioned pathogens. Summary of the invention

[0007] In view of this, in a first aspect, the present invention provides a composition for detecting urinary tract infection-related pathogens and their drug resistance, comprising upstream and downstream primers and probes for detecting at least three of the following seven targets:

[0008] Upstream primers, downstream primers and probes for detecting Citrobacter freundii as shown in SEQ ID NOs: 1 to 3;

[0009] Upstream primers, downstream primers and probes for detecting Proteus vulgaris as shown in SEQ ID NOs: 4 to 6; and

[0010] Upstream primers, downstream primers and probes for detecting Providencia as shown in SEQ ID NOs: 7 to 9;

[0011] Upstream primers, downstream primers and probes for detecting the AmpC gene as shown in SEQ ID NOs: 10 to 12;

[0012] Upstream primers, downstream primers and probes for detecting TEM genes as shown in SEQ ID NOs: 13 to 15;

[0013] Upstream primers, downstream primers and probes for detecting NDM-1 gene as shown in SEQ ID NOs: 16 to 18; or

[0014] The upstream primers, downstream primers and probe for detecting the NDM-2 gene are shown in SEQ ID NOs: 19-21.

[0015] In some specific embodiments, the present invention provides a composition for detecting urinary tract infection-related pathogens and their drug resistance, comprising:

[0016] Upstream primers, downstream primers and probes for detecting Citrobacter freundii as shown in SEQ ID NOs: 1 to 3;

[0017] Upstream primers, downstream primers and probes for detecting Proteus vulgaris as shown in SEQ ID NOs: 4 to 6; and

[0018] The upstream primers, downstream primers and probe for detecting Providencia are shown in SEQ ID NOs: 7-9.

[0019] Furthermore, the composition also includes upstream and downstream primers and probes for detecting at least one of the following four targets:

[0020] Upstream primers, downstream primers and probes for detecting the AmpC gene as shown in SEQ ID NOs: 10 to 12;

[0021] Upstream primers, downstream primers and probes for detecting TEM genes as shown in SEQ ID NOs: 13 to 15;

[0022] Upstream primers, downstream primers and probes for detecting NDM-1 gene as shown in SEQ ID NOs: 16 to 18; or

[0023] The upstream primers, downstream primers and probe for detecting the NDM-2 gene are shown in SEQ ID NOs: 19-21.

[0024] Furthermore, the composition of the present invention includes upstream and downstream primers and probes for at least 4, 5, 6, or 7 of the 7 targets as described above.

[0025] The combined detection composition provided by the present invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different targets by detecting targets on different pathogens, thereby simultaneously realizing the detection and differentiation of Citrobacter freundii, Proteus vulgaris, Providencia, and AmpC genes, TEM genes, and NDM-1 and NDM-2 genes in a single-tube reaction system, so as to provide a targeted strategy for subsequent treatment. The composition of the present invention has a higher detection sensitivity of 200 copies / mL, good specificity, and more accurate detection, so that clinicians can provide targeted strategies for the prevention and treatment of infections caused by the above-mentioned pathogens.

[0026] Furthermore, the present invention provides a composition for detecting urinary tract infection-related pathogens and their drug resistance, comprising:

[0027] Upstream primers, downstream primers and probes for detecting Citrobacter freundii as shown in SEQ ID NOs: 1 to 3;

[0028] Upstream primers, downstream primers and probes for detecting Proteus vulgaris as shown in SEQ ID NOs: 4 to 6;

[0029] Upstream primers, downstream primers and probes for detecting Providencia as shown in SEQ ID NOs: 7 to 9;

[0030] Upstream primers, downstream primers and probes for detecting the AmpC gene as shown in SEQ ID NOs: 10 to 12;

[0031] Upstream primers, downstream primers and probes for detecting TEM genes as shown in SEQ ID NOs: 13 to 15;

[0032] Upstream primers, downstream primers and probes for detecting NDM-1 gene as shown in SEQ ID NOs: 16 to 18; and

[0033] The upstream primers, downstream primers and probe for detecting the NDM-2 gene are shown in SEQ ID NOs: 19-21.

[0034] Furthermore, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

[0035] In some specific embodiments, an exogenous internal standard is added to the sample, and further, the internal standard is a human internal standard gene.

[0036] In a specific embodiment, the internal standard is RNase P or NADPH.

[0037] Furthermore, the fluorescent groups of the probes used in the composition of the present invention to detect different targets are different from each other and do not interfere with each other.

[0038] 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, ATTO425, Quasar705, 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.

[0039] In some specific embodiments, the fluorescent reporter group of the probe for detecting Citrobacter freundii is ATTO425; the fluorescent reporter group of the probe for detecting Proteus vulgaris is FAM; the fluorescent reporter group of the probe for detecting Providencia is HEX; the fluorescent reporter group of the probe for detecting the AmpC gene is ROX; the fluorescent reporter group of the probe for detecting the TEM gene is CY5; and the fluorescent reporter group of the probe for detecting the NDM-1 or 2 gene is QUASAR 705.

[0040] Further, in some embodiments, the composition of the present invention may include one or more of the above primer and probe pairs. In the present invention, "set" refers to upstream and downstream primers and probes that match each other for detecting a target.

[0041] The composition of the present invention can be combined into any combination of detection corresponding to the 7 targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the primers and probe pairs corresponding to the targets. These combinations are all included in the present invention.

[0042] For example, it may include any 6 of the above 7 groups of primers and probes, it may include any 5 of the above 7 groups of primers and probes, it may include any 4 of the above 7 groups of primers and probes, it may include any 3 of the above 7 groups of primers and probes, it may include any 2 of the above 7 groups of primers and probes, and it may include any 1 of the above 7 groups of primers and probes.

[0043] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0044] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0045] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.

[0046] In a specific embodiment, the 3' end of the probe is BHQ1.

[0047] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0048] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0049] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0050] 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 detecting urinary tract infection-related pathogens and their drug resistance, wherein the target is Citrobacter freundii, Proteus vulgaris, Providencia, one or more of the AmpC gene, TEM gene, or NDM-1 or 2 gene.

[0051] In a third aspect, the present invention provides a kit for detecting urinary tract infection-related pathogens and their drug resistance, wherein the kit comprises the composition of the present invention as described above.

[0052] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0053] In a specific embodiment, the negative control is at least one of DEPC H2O and normal saline; the positive control is a fragment plasmid or positive strain of Citrobacter freundii, Proteus vulgaris, Providencia, and at least one of a fragment plasmid of AmpC gene, TEM gene, or NDM-1 or 2 gene.

[0054] Furthermore, the kit also includes dNTP(U)s, PCR buffer and Mg2+ At least one of .

[0055] Furthermore, the kit also includes at least one of DNA polymerase and uracil glycosylase (UNG).

[0056] Furthermore, the kit also includes at least one of a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0057] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP, dUTP, uracil glycosylase (UNG), DNA polymerase, UNG enzyme, PCR buffer and Mg 2+ At least one of .

[0058] Furthermore, the concentration of the DNA polymerase is 5 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0059] In a specific embodiment, the kit of the present invention comprises Taq enzyme, UDG enzyme, Mg 2+ , dNTP(U)s, primers, probes and PCR buffer.

[0060] The main components of PCR buffer include Tris-HCl, NaCl, NH4Cl, and Tween 20.

[0061] In some specific embodiments, the total volume of the reactants in a single PCR reaction tube is 20 μL to 60 μL.

[0062] 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.

[0063] In a fourth aspect, a method for detecting urinary tract infection-related pathogens and their drug resistance is provided, the method comprising the following steps:

[0064] 1) Extracting nucleic acid from the sample to be tested;

[0065] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent PCR detection on the nucleic acid obtained in step 1);

[0066] 3) Obtain and analyze the results.

[0067] In the present invention, the sample used for detection may be urine or the like, but is not limited thereto.

[0068] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0069] UNG enzyme reaction, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; Taq enzyme activation, temperature is 90-99℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-99℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0070] Furthermore, the method for detecting urinary tract infection-related pathogens and their drug resistance is for non-diagnostic purposes.

[0071] In a specific embodiment, a composition is provided for preparing a reagent for detecting urinary tract infection-related pathogens and their drug resistance, wherein the detection comprises the following steps:

[0072] 1) Extracting nucleic acid from the sample to be tested;

[0073] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent PCR detection on the nucleic acid obtained in step 1);

[0074] 3) Obtain and analyze the results.

[0075] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0076] UNG enzyme reaction, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; Taq enzyme activation, temperature is 90-99℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-99℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0077] In this article, the term "non-diagnostic purpose" means that it is not intended to obtain information on whether an individual is infected with the above pathogens and suffers from the infection. For example, the method can detect whether the above pathogens are present in the environment and culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figures 1 to 4 The results of testing different samples for the composition of the present invention are shown in FIG.

[0079] Figure 5 This is a sensitivity result diagram of the composition of the present invention;

[0080] Figure 6 It is a specific result diagram of the composition of the present invention;

[0081] Figures 7-8 This is a graph showing the test results of different compositions of the comparative examples of the present invention. DETAILED DESCRIPTION

[0082] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0083] Example 1: Primers and probes used in the present invention

[0084] The primers and probes used in the present invention are shown in Table 1 below:

[0085] Table 1

[0086]

[0087]

[0088] Among them, the fluorescent reporter group of the probe for detecting Citrobacter freundii is ATTO 425; the fluorescent reporter group of the probe for detecting Proteus vulgaris is FAM; the fluorescent reporter group of the probe for detecting Providencia is HEX; the fluorescent reporter group of the probe for detecting AmpC gene is ROX; the fluorescent reporter group of the probe for detecting TEM gene is CY5; the fluorescent reporter group of the probe for detecting NDM-1 or 2 gene is QUASAR 705.

[0089] Example 2: Method for detecting pathogens

[0090] Reagent preparation

[0091] Take the corresponding amount of PCR reaction solution and enzyme mixture (PCR reaction solution 38 μL / person + enzyme mixture 2 μL / person) in proportion, and mix them thoroughly to form a PCR mixture.

[0092] 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.

[0093] Sample handling and loading

[0094] Take 300 μL of the sample to be tested, negative control, and positive control into a 1.5 mL centrifuge tube, and use the nucleic acid extraction or purification reagent of Shengxiang Biotechnology Co., Ltd. according to its instructions to extract nucleic acid.

[0095] 10 μL of the treated samples, negative controls and positive controls were pipetted into reaction tubes, 40 μL of PCR mixture was added to each reaction tube and the tube cap was covered. The real-time fluorescence PCR reaction system was configured according to Table 2 below.

[0096] Table 2

[0097] Components The volume of each reaction PCR buffer* 33.8μL dNTPs(100mM) 0.5μL <![CDATA[1mol / L MgCl2]]> 0.2μL Primer ((50pmol / μL)) 0.2μL / strip Probe ((50pmol / μL)) 0.1μL / strip

[0098] PCR amplification

[0099] PCR amplification was performed on PCR instruments such as ABI 7500 fluorescence quantitative PCR instrument and SLAN-96P fully automatic medical PCR analysis system according to certain temperature and time setting programs. PCR amplification was performed according to the temperature and time setting programs shown in Table 3.

[0100] Table 3

[0101]

[0102] Result analysis and judgment

[0103] If ATTO 425, FAM, HEX, CY5, ROX and Quasar705 channels have obvious S-shaped amplification curves and Ct values ​​≤ 38, it is judged as positive; if ATTO 425, FAM, HEX, ROX and Quasar705 channels have no amplification curve (No Ct) or Ct values ​​> 38, and CY5 ≤ 38, it is judged as negative; if ATTO 425, FAM, HEX, ROX and Quasar705 channels have no amplification curve (No Ct) or Ct values ​​> 38, and CY5 > 38, the result is unreliable and needs to be retested. The details are shown in Table 4 below.

[0104] Table 4

[0105]

[0106] Example 3: Test results of the test samples of the composition of the present invention

[0107] The primers and probes shown in Example 1 were used to perform PCR detection on different mixed samples of Citrobacter freundii, Proteus vulgaris, Providencia, AmpC gene, TEM gene and NDM gene on a SLAN-96P real-time fluorescence quantitative PCR instrument according to the method of Example 2. The results showed that the composition of the present invention can detect and distinguish any combination of the above-mentioned related targets. For example, the composition of the present invention detects:

[0108] Citrobacter freundii, Proteus vulgaris, Providencia Figure 1 );

[0109] Citrobacter freundii, Providencia, Providencia, AmpC gene, TEM gene ( Figure 2 );

[0110] AmpC gene, Proteus vulgaris, TEM gene, NDM gene ( Figure 3 );

[0111] Citrobacter freundii, Proteus vulgaris, Providencia, AmpC gene, TEM gene, NDM gene ( Figure 4 ).

[0112] These figures can prove that the present invention is a 6-plex PCR that can amplify the above-mentioned 7 targets at one time, and can use a 6-channel PCR instrument to distinguish and design primers and probes to prevent missed detection of the NDM gene. It also proves that any corresponding pair of combinations of the composition of the present invention can detect the corresponding combination of any target.

[0113] Example 4: Sensitivity of the composition of the present invention

[0114] The LOD (sensitivity) of each pathogen was tested, and the results are shown in Table 5 and Figure 5 As shown, the test results show that the composition can still accurately detect samples as low as 200 copies / mL in each channel.

[0115] Table 5

[0116]

[0117] Example 5. Specificity of the composition of the present invention

[0118] The composition of the present invention has no cross-reaction with pathogens related to urinary tract infection and other pathogens with similar drug resistance (Staphylococcus aureus, Escherichia coli, Proteus, Neisseria gonorrhoeae, Mycoplasma, Chlamydia, Klebsiella, Citrobacter, Enterococcus faecalis and Pseudomonas aeruginosa). Figure 6 As shown, it is shown that the composition of the present invention has good specificity.

[0119] Example 6: Anti-interference performance of the composition of the present invention

[0120] The composition of the present invention 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), co-trimoxazole (50 μg / mL), clindamycin (50 μg / mL), heme (10 μg / mL), hemoglobin (10%), etc. at certain concentrations had no obvious 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.

[0121] Table 6

[0122]

[0123] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0124] 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. To ensure the conservatism of the design (conservatism is crucial to the accuracy of the test), and to consider the mutual interference between different primers and probes, the primers and probes need to be carefully designed.

[0125] Therefore, the inventors also designed some other primers and probes to form different detection systems (the sequences are shown in Table 7, and another set of primers and probes for detecting AmpC genes with better detection effects alone were used to replace the primers and probes for AmpC genes in the composition of the present invention), which were also used to detect the above pathogens. Specific detection results are shown in the following examples. Figure 7 As shown, from Figure 7 It can be seen that the detection performance of the AmpC gene is reduced, the Ct value is delayed, and the fluorescence increment decreases. It is worth noting that the single detection effect of the AmpC gene before and after replacement is equivalent, such as Figure 8 shown.

[0126] Table 7

[0127]

Claims

1. A composition for detecting urinary tract infection-related pathogens and their drug resistance, comprising upstream and downstream primers and probes for detecting at least three of the following seven targets: Upstream primers, downstream primers and probes for detecting Citrobacter freundii as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Proteus vulgaris as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting Providencia as shown in SEQ ID NOs: 7 to 9; Upstream primers, downstream primers and probes for detecting the AmpC gene as shown in SEQ ID NOs: 10 to 12; Upstream primers, downstream primers and probes for detecting TEM genes as shown in SEQ ID NOs: 13 to 15; Upstream primers, downstream primers and probes for detecting NDM-1 gene as shown in SEQ ID NOs: 16 to 18; or The upstream primers, downstream primers and probe for detecting the NDM-2 gene are shown in SEQ ID NOs: 19-21.

2. The composition according to claim 2, characterized in that The composition includes upstream and downstream primers and probes for at least 4, 5, 6, or 7 of the 7 targets as described above.

3. The composition according to claim 2, characterized in that The composition comprises: Upstream primers, downstream primers and probes for detecting Citrobacter freundii as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Proteus vulgaris as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting Providencia as shown in SEQ ID NOs: 7 to 9; Upstream primers, downstream primers and probes for detecting the AmpC gene as shown in SEQ ID NOs: 10 to 12; Upstream primers, downstream primers and probes for detecting TEM genes as shown in SEQ ID NOs: 13 to 15; Upstream primers, downstream primers and probes for detecting NDM-1 gene as shown in SEQ ID NOs: 16 to 18; and The upstream primers, downstream primers and probe for detecting the NDM-2 gene are shown in SEQ ID NOs: 19-21.

4. The composition according to claim 3, characterized in that The fluorescent reporter group of the probe for detecting Citrobacter freundii is ATTO 425; the fluorescent reporter group of the probe for detecting Proteus vulgaris is FAM; the fluorescent reporter group of the probe for detecting Providencia is HEX; the fluorescent reporter group of the probe for detecting AmpC gene is ROX; the fluorescent reporter group of the probe for detecting TEM gene is CY5; the fluorescent reporter group of the probe for detecting NDM-1 or 2 gene is QUASAR 705.

5. The composition according to claim 1, characterized in that The composition comprises an upstream primer, a downstream primer and a probe for detecting an internal standard.

6. The composition according to claim 1, characterized in that The components of the composition are present in admixture.

7. Use of the composition according to any one of claims 1 to 6 in preparing a kit for detecting pathogens associated with urinary tract infection and their drug resistance, wherein: The target is one or more of Citrobacter freundii, Proteus vulgaris, Providencia, AmpC gene, TEM gene, or NDM-1 or 2 gene.

8. A kit for detecting pathogens associated with urinary tract infection and their drug resistance, the kit comprising the composition according to any one of claims 1 to 6.

9. The kit according to claim 8, characterized in that The kit further comprises: a nucleic acid releasing reagent, a nucleic acid extracting reagent, a DNA polymerase, a UNG enzyme, dNTP, dUTP, a UNG enzyme, a PCR buffer, or a Mg 2+ At least one of .

10. Use of a composition for preparing a reagent for detecting pathogens associated with urinary tract infection and their drug resistance, the detection comprising the following steps: 1) Extracting nucleic acid from the sample to be tested; 2) performing fluorescent PCR detection on the nucleic acid obtained in step 1) using the composition according to any one of claims 1 to 6 or the kit according to any one of claims 8 to 9; 3) Obtain and analyze the results.

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