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

Abstract

The invention belongs to the field of molecular biology detection, particularly relates to detection of urinary tract infection related pathogens, and more particularly relates to detection of at least four of ureazonibacterium urealyticum, corynebacterium urealyticum, citrobacter kirchkii, citrobacter freundii and staphylococcus saprophyticus. The composition provided by the invention has the advantages of higher detection sensitivity (up to 500 copies / mL), good specificity and more accurate detection, provides a more sufficient basis for rapid diagnosis and exclusion of infection of different pathogens for clinicians, shortens the time for the clinicians to diagnose the condition of a patient, and accelerates the implementation of treatment measures on the patient.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to the detection of pathogens related to urinary tract infection, and more specifically, to the detection of Ureaerococcosis, Ureaplasma urealyticum, Citrobacter krusei, Citrobacter freundii, and Staphylococcus saprophyticus. Background Art

[0002] Aerococcus urinae is a Gram-positive, catalase-negative coccus that grows in clusters. Aerococcus urinae is a rarely reported pathogen that can be isolated from the urine of patients with UTIs presenting with a variety of clinical conditions, including urinary tract infection (UTI), urge urinary incontinence (UUI), and overactive bladder (OAB). Due to the difficulties in biochemical identification of Aerococcus urinae in clinical microbiology laboratories, Aerococcus urinae is easily mischaracterized as streptococci, enterococci, or staphylococci. Aerococcus urinae is highly resistant to many sulfonamides and other antibiotics used to treat UTIs and, if undiagnosed or misdiagnosed, can cause invasive and life-threatening bacteremia, sepsis, or soft tissue infections.

[0003] Corynebacterium urealyticum is a slow-growing, lipophilic, multidrug-resistant, urease-positive Gram-positive bacterium. It is reported to be an opportunistic nosocomial pathogen causing a variety of diseases including, but not limited to, cystitis, pyelonephritis, and bacteremia. Most strains of Corynebacterium urealyticum are highly resistant to a wide range of antibiotics including aminoglycosides, beta-lactams, and macrolides.

[0004] Citrobacter koseri is a Gram-negative bacterium that is oxidase-negative and catalase-positive. It is one of the causes of sepsis and meningitis with central nervous system (CNS) abscesses in infants and immunocompromised individuals, and can cause respiratory and urinary tract infections and skin infections in adults. It is a conditional pathogen that is highly resistant to a variety of antibiotics, including aminoglycosides and quinolones, while quinolones (especially ciprofloxacin and levofloxacin) are the most commonly used antibiotics for the treatment of urinary tract infections.

[0005] Citrobacter freundii is a Gram-negative, facultative anaerobic, non-spore-forming, rod-shaped bacillus, and the most infectious species in the genus Citrobacter. It is involved in a range of nosocomial infectious diseases, including bloodstream infections (BSI), respiratory tract infections (RTI), biliary tract infections (BTI), and urinary tract infections (UTI). Multidrug-resistant Citrobacter freundii has developed resistance to multiple antibiotics including extended-spectrum β-lactamases, carbapenemases, cephalosporins, and quinolones. This has become a major challenge for the healthcare sector.

[0006] Staphylococcus saprophyticus is a gram-positive, coagulase-negative, non-hemolytic coccus that is the second most common cause of community-acquired urinary tract infections, after Escherichia coli. Approximately 20-40% of all urinary tract infections in middle-aged women are caused by Staphylococcus saprophyticus. It can also lead to complications such as acute pyelonephritis, urethritis, epididymitis, and prostatitis.

[0007] The above pathogens are difficult to detect in traditional tests, and they all cause urinary tract infections with similar symptoms. For infected patients, early diagnosis and treatment are needed, and the progression of the disease should be controlled in a targeted and timely manner.

[0008] Therefore, there is a need in the art for a product that can simply and quickly detect the above-mentioned pathogens, and has good sensitivity and specificity, and can detect and distinguish pathogenic microorganisms that cause infection, so as to provide targeted strategies for the prevention and treatment of the above-mentioned infections. Summary of the invention

[0009] In view of this, in a first aspect, the present invention provides a composition for detecting pathogens associated with urinary tract infection, comprising upstream and downstream primers and probes for detecting at least 4 of the following related pathogens:

[0010] Upstream primers, downstream primers and probes for detecting Ureaplasma as shown in SEQ ID NOs: 1 to 3;

[0011] Upstream primers, downstream primers and probes for detecting Corynebacterium urealyticum as shown in SEQ ID NOs: 4 to 6;

[0012] Upstream primers, downstream primers and probes for detecting Citrobacter krusei as shown in SEQ ID NOs: 7 to 9;

[0013] The upstream primers, downstream primers and probe for detecting Citrobacter freundii as shown in SEQ ID NOs: 10 to 12; or

[0014] The upstream primers, downstream primers and probe for detecting Staphylococcus saprophyticus are shown in SEQ ID NOs: 13-15.

[0015] The combined detection composition provided by the present invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby simultaneously realizing the detection and differentiation of at least 4 of Ureaplasma ureaphylla, Ureaplasma urealyticum, Citrobacter krusei, and Citrobacter freundii 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 sensitivity of detection, reaching 500 copies / mL, good specificity, and more accurate detection, providing a relatively sufficient basis for rapid diagnosis and exclusion of different pathogen infections for clinicians, shortening the time for clinicians to diagnose the patient's condition, and accelerating the implementation of treatment measures for patients.

[0016] Furthermore, the present invention provides a composition for detecting pathogens associated with urinary tract infection, comprising:

[0017] Upstream primers, downstream primers and probes for detecting Ureaplasma as shown in SEQ ID NOs: 1 to 3;

[0018] Upstream primers, downstream primers and probes for detecting Corynebacterium urealyticum as shown in SEQ ID NOs: 4 to 6;

[0019] Upstream primers, downstream primers and probes for detecting Citrobacter krusei as shown in SEQ ID NOs: 7 to 9;

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

[0021] The upstream primers, downstream primers and probe for detecting Staphylococcus saprophyticus are shown in SEQ ID NOs: 13-15.

[0022] Furthermore, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

[0023] In some specific embodiments, an exogenous internal standard is added to the sample, and further, the internal standard is a human internal standard gene.

[0024] In a specific embodiment, the internal standard is RNase P or NADPH.

[0025] Furthermore, the fluorescent groups of the probes used in the composition of the present invention to detect different pathogens are different from each other and do not interfere with each other.

[0026] 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.

[0027] In some specific embodiments, the fluorescent reporter group of the probe for detecting Ureaplasma urealyticum is FAM; the fluorescent reporter group of the probe for detecting Ureaplasma urealyticum is HEX; the fluorescent reporter group of the probe for detecting Citrobacter krusei is ROX; the fluorescent reporter group of the probe for detecting Citrobacter freundii is CY5; and the fluorescent reporter group of the probe for detecting Staphylococcus saprophyticus is Quasar 705.

[0028] 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.

[0029] The composition of the present invention can be combined into any combination of detection corresponding to 5 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.

[0030] For example, it may include any 4 of the above 5 sets of primers and probes, any 3 of the above 5 sets of primers and probes, any 2 of the above 5 sets of primers and probes, or any 1 of the above 5 sets of primers and probes.

[0031] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0032] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0033] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.

[0034] In a specific embodiment, the 3' end of the probe is BHQ1.

[0035] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0036] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0037] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0038] 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 pathogens related to urinary tract infection, wherein the pathogen is one or more of Citrobacter freundii, Ureaerococcosis, Ureaplasma urealyticum, Citrobacter krusei, or Staphylococcus saprophyticus.

[0039] In a third aspect, the present invention provides a kit for detecting pathogens associated with urinary tract infection, wherein the kit comprises the composition of the present invention as described above.

[0040] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0041] In a specific embodiment, the negative control is at least one of DEPC H2O and normal saline; the positive control is at least one of a fragment plasmid or a positive strain of Citrobacter freundii, Ureaplasma urealyticum, Corynebacterium urealyticum, Citrobacter kluyveri, and Staphylococcus saprophyticus.

[0042] Furthermore, the kit also includes dNTP(U)s, PCR buffer and Mg 2+ At least one of .

[0043] Furthermore, the kit also includes at least one of DNA polymerase and uracil glycosylase (UNG).

[0044] Furthermore, the kit also includes at least one of a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0045] 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 .

[0046] Furthermore, the concentration of the DNA polymerase is 5 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0047] 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.

[0048] The main components of PCR buffer include Tris-HCl, NaCl, NH4Cl, and Tween 20.

[0049] In some specific embodiments, the total volume of the reactants in a single PCR reaction tube is 20 μL to 60 μL.

[0050] 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.

[0051] In a fourth aspect, a method for detecting pathogens associated with urinary tract infection is provided, the method comprising the following steps:

[0052] 1) Extracting nucleic acid from the sample to be tested;

[0053] 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);

[0054] 3) Obtain and analyze the results.

[0055] In the present invention, the sample used for detection may be urine or the like, but is not limited thereto.

[0056] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0057] UNG decontamination, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; pre-denaturation, 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 collection.

[0058] Furthermore, the method for detecting urinary tract infection-related pathogens is for non-diagnostic purposes.

[0059] In a specific embodiment, a composition is provided for preparing a reagent for detecting pathogens associated with urinary tract infection, wherein the detection comprises the following steps:

[0060] 1) Extracting nucleic acid from the sample to be tested;

[0061] 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);

[0062] 3) Obtain and analyze the results.

[0063] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0064] UNG decontamination, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; pre-denaturation, 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 collection.

[0065] 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

[0066] Figures 1 to 3 The results of testing different samples for the composition of the present invention are shown in FIG.

[0067] Figures 4 to 8 is a sensitivity result diagram of the composition of the present invention (Ureaerostomella, Corynebacterium urealyticum, Citrobacter krusei, Citrobacter freundii, and Staphylococcus saprophyticus, respectively);

[0068] Fig. 9 It is a specific result diagram of the composition of the present invention;

[0069] Figures 10 to 14 This is a graph showing the precision results of the composition of the present invention (Ureaerostomella, Corynebacterium urealyticum, Citrobacter krusei, Citrobacter freundii, and Staphylococcus saprophyticus, respectively);

[0070] Figures 15-16 This is a graph showing the test results of different compositions of the comparative examples of the present invention. DETAILED DESCRIPTION

[0071] 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.

[0072] Example 1: Primers and probes used in the present invention

[0073] The primers and probes used in the present invention are shown in Table 1 below:

[0074] Table 1

[0075]

[0076] Among them, the fluorescent reporter group of the Ureaplasma urealyticum probe is FAM; the fluorescent reporter group of the Ureaplasma urealyticum probe is HEX; the fluorescent reporter group of the Citrobacter kluyveri probe is ROX; the fluorescent reporter group of the Citrobacter freundii probe is CY5; and the fluorescent reporter group of the probe for detecting Staphylococcus saprophyticus is Quasar705.

[0077] Example 2: Method for detecting pathogens

[0078] Reagent preparation

[0079] Take out each component in the box, place it at room temperature, wait for the temperature to equilibrate to room temperature, mix well and centrifuge for a short time before use.

[0080] According to the number of samples to be tested, negative controls, and positive controls, take the corresponding amount of components in proportion (PCR reaction solution 38 μL / person + enzyme mixture 2 μL / person), mix them thoroughly to form a PCR mixture, and centrifuge it for standby use.

[0081] Transfer the above prepared reagents to the sample processing area for later use.

[0082] Sample handling and loading

[0083] The sample tested in the present invention is urine. The viral nucleic acid is extracted by magnetic bead method, and the following operations are performed in the sample processing room:

[0084] Take a 1.5mL cryotube and add glass beads. Add 300uL of the treated sample and 300uLTE to the 1.5mL cryotube containing mixed glass beads and grind using a grinder. When the grinding is completed, take out the cryotube and centrifuge it instantly. Take out the supernatant liquid as the sample to be tested for later use.

[0085] Take a number of 1.5mL centrifuge tubes according to the number of samples to be tested, and add 300μL of the sample to be tested to each tube;

[0086] Add 500 μL of extraction solution 1 and 50 μL of proteinase K-magnetic bead mixture; cover the tube, shake and mix for 30 seconds, and heat at 60°C for 10 minutes.

[0087] Let stand at room temperature for 1 min, centrifuge at low speed, place the centrifuge tube on the magnetic separator, and slowly discard the waste liquid after 5 min (*Be careful not to touch the magnetic beads adsorbed on the inner side of the tube wall);

[0088] Add 600 μL of washing solution 1, shake and mix for 30 seconds, centrifuge at low speed, and place the centrifuge tube in a magnetic separator. Magnetic suction for 3 minutes, completely aspirate and discard the liquid.

[0089] Add 600 μL of washing solution 2, shake and mix for 30 seconds, centrifuge at low speed, and place the centrifuge tube in a magnetic separator. Magnetic suction for 3 minutes, completely aspirate and discard the liquid.

[0090] Place the centrifuge tube in a centrifuge and centrifuge it at low speed for a short time, then place the centrifuge tube in a magnetic separator again. Magnetic suction for 3 minutes to completely absorb the liquid at the bottom of the tube.

[0091] Add 30-100 μL of elution buffer S (80 μL is recommended), shake and mix for 30 seconds to elute the magnetic beads on the wall of the centrifuge tube to the bottom of the tube, and let it stand at room temperature for 3 minutes; centrifuge at a low speed, place the centrifuge tube on the magnetic separator again for 3 minutes, and then transfer the eluted nucleic acid to a clean 1.5 mL centrifuge tube.

[0092] 10 μL of the treated samples, positive controls and negative controls were added to the corresponding 0.2 mL PCR reaction tubes, 40 μL of PCR mixture was added to each tube, and the tube cap was closed. The real-time fluorescence PCR reaction system was configured according to Table 2 below.

[0093] Table 2

[0094]

[0095] PCR amplification

[0096] 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.

[0097] Table 3

[0098]

[0099] Result analysis and judgment

[0100] After the reaction is completed, the results are automatically saved, and the amplification curves of the detection target and the internal standard are analyzed separately. According to the analyzed image, adjust the Start value, End value and Threshold value of the Baseline (the user can adjust it according to the actual situation, the Start value can be set between 3 and 15, the End value can be set between 5 and 20, and adjust the amplification curve of the negative control to make it flat or below the threshold line), click Analyze to analyze, make all parameters meet the requirements of the following "Quality Control", and then record the qualitative results in the Plate window.

[0101] Quality Control

[0102] Negative control: FAM, HEX, ROX, CY5, and Quasar 705 channel Ct>40 or no Ct value (NoCt);

[0103] Positive control: FAM, HEX, ROX, CY5 and Quasar 705 channels all had Ct≤35;

[0104] The above requirements must be met simultaneously in the same experiment, otherwise, the experiment will be invalid and must be repeated.

[0105] Positive judgment value

[0106] The Ct reference value of the target gene detected by this kit was determined to be 40 through reference value research.

[0107] Interpretation of Test Results

[0108] The evaluation of the test results of clinical specimens should be performed after the positive and negative controls have been tested and determined to be valid and acceptable. If the quality control is invalid, the patient's results cannot be interpreted. The following table describes the interpretation of the results regarding the use of the above quality control products. The end user needs to check the fluorescence curves before the final interpretation. For weak positive samples, all curves are typical S-shaped amplification curves or no plateau phase (38≤Ct≤40). The details are shown in Table 4 below.

[0109] Table 4

[0110]

[0111]

[0112] Example 3: Test results of the test samples of the composition of the present invention

[0113] The primers and probes shown in Example 1 were used to perform PCR detection on a mixed sample of Ureaplasma urealyticum, Corynebacterium urealyticum, Citrobacter krusei, and Citrobacter freundii 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 pathogens. For example, the composition of the present invention detects:

[0114] Ureaplasma urealyticum, Citrobacter kremeri, Citrobacter freundii, Staphylococcus saprophyticus ( Figure 1 );

[0115] Ureaplasma, Citrobacter kremeri, Citrobacter freundii, Staphylococcus saprophyticus ( Figure 2 );

[0116] Ureaplasma, Corynebacterium urealyticum, Citrobacter kremeri, Citrobacter freundii, Staphylococcus saprophyticus ( Figure 3 ).

[0117] These figures can prove that the present invention is a 5-plex PCR that can amplify the above-mentioned 5 targets at one time and can be distinguished using a 5-channel PCR instrument. It also proves that any corresponding pair combination of the composition of the present invention can detect any corresponding combination of pathogens.

[0118] Example 4: Sensitivity of the composition of the present invention

[0119] The composition of Example 1 of the present invention was used to detect samples with a concentration as low as 500 copies / mL. Figures 4 to 8 As shown, it is shown that each channel can still accurately detect samples as low as 500 copies / mL, with a detection rate of 100%, indicating that the sensitivity of the composition of the present invention is 500 copies / mL.

[0120] Example 5. Specificity of the composition of the present invention

[0121] The composition of the present invention has no cross-reaction with other pathogens similar to urinary tract infection-related pathogens (Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Proteus mirabilis, Mobiluncus fragilis, Bacteroides fragilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Candida albicans, Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis). Fig. 9 As shown, it is shown that the composition of the present invention has good specificity.

[0122] Example 6: Precision of the composition of the present invention

[0123] The system of the present invention dilutes the positive sample to a medium positive concentration level (5.0×10 3 The precision of the test was determined by repeating the test 10 times for each sample. The detection rate of the reference product was 100%, and the coefficient of variation (CV) of the Ct value was less than 5%, indicating that the composition of the present invention had good detection precision within and between batches. The results are shown in FIG. Figures 10 to 14 shown.

[0124] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0125] 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.

[0126] Therefore, the inventors also designed some other primers and probes to form different detection systems (the sequences are shown in Table 5, and another set of primers and probes for detecting Citrobacter freundii (combination 2) was used to replace the primers and probes for detecting Citrobacter freundii in the composition of the present invention (combination 1)), which were also used to detect the above pathogens. Specific detection results are shown in the following examples. Figures 15-16 As shown, from Fig.15 It can be seen that the primer and probe set of Citrobacter freundii (combination 2) used in the comparative example is better than the primer and probe set of Citrobacter freundii (combination 1) in single target amplification. However, after combining it with the other 4 primer and probe sets of the composition of the present invention, it was found that ( Fig.16 ), the Ct value of the test is low and some targets do not produce lines, so the overall detection effect is poor.

[0127] Table 5

[0128]

Claims

1. A composition for detecting pathogens associated with urinary tract infection, comprising upstream and downstream primers and probes for detecting at least 4 of the following related pathogens: Upstream primers, downstream primers and probes for detecting Ureaplasma as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Corynebacterium urealyticum as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting Citrobacter krusei as shown in SEQ ID NOs: 7 to 9; The upstream primers, downstream primers and probe for detecting Citrobacter freundii as shown in SEQ ID NOs: 10 to 12; or The upstream primers, downstream primers and probe for detecting Staphylococcus saprophyticus are shown in SEQ ID NOs: 13-15.

2. The composition according to claim 1, characterized in that The composition comprises: Upstream primers, downstream primers and probes for detecting Ureaplasma as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Corynebacterium urealyticum as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting Citrobacter krusei as shown in SEQ ID NOs: 7 to 9; Upstream primers, downstream primers and probes for detecting Citrobacter freundii as shown in SEQ ID NOs: 10 to 12; and The upstream primers, downstream primers and probe for detecting Staphylococcus saprophyticus are shown in SEQ ID NOs: 13-15.

3. The composition according to claim 2, characterized in that The fluorescent reporter group of the probe for detecting Ureaplasma urealyticum is FAM; the fluorescent reporter group of the probe for detecting Corynebacterium urealyticum is HEX; the fluorescent reporter group of the probe for detecting Citrobacter krusei is ROX; the fluorescent reporter group of the probe for detecting Citrobacter freundii is CY5; and the fluorescent reporter group of the probe for detecting Staphylococcus saprophyticus is Quasar 705.

4. The composition according to any one of claims 1 to 3, characterized in that The composition also includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

5. The composition according to claim 4, characterized in that The components of the composition are present in admixture.

6. Use of the composition according to any one of claims 1 to 5 in preparing a kit for detecting pathogens associated with urinary tract infection, wherein: The pathogen is one or more of Citrobacter freundii, Ureaplasma urealyticum, Corynebacterium urealyticum, Citrobacter kluyveri, or Staphylococcus saprophyticus.

7. A kit for detecting pathogens associated with urinary tract infection, the kit comprising the composition according to any one of claims 1 to 5.

8. The kit according to claim 7, characterized in that The kit also includes a negative quality control product and a positive quality control product.

9. The kit according to claim 7 or 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, 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 5 or the kit according to any one of claims 7 to 9; 3) Obtain and analyze the results.

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