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

Abstract

The invention belongs to the technical field of molecular biology in-vitro nucleic acid detection, and particularly relates to a composition and method for detecting staphylococcus aureus, enterococcus faecalis and enterococcus faecium as well as detecting and distinguishing a tetracycline drug-resistant gene Tet (M), a tetracycline drug-resistant gene Tet (L), a vancomycin drug-resistant gene vanA and a vancomycin drug-resistant gene vanB while detecting and distinguishing staphylococcus aureus, enterococcus faecalis and enterococcus faecium, and application of the composition and method. According to the combined detection composition provided by the invention, different pathogens and drug-resistant genes are detected by detecting targets on different pathogens and drug-resistant genes by utilizing a multiple fluorescent PCR analysis method, so that three pathogen target genes and four drug-resistant gene segments can be rapidly detected in one tube of reaction liquid, and the combined detection composition has the advantages of higher detection sensitivity, higher sensitivity, higher sensitivity, higher sensitivity and the like. The copy / mL reaches 200 copies / mL. In addition, 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 technical field of in vitro nucleic acid detection in molecular biology, and specifically to the detection of multiple urinary tract infection pathogens and drug-resistant genes; more specifically, it relates to the detection of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, and tetracycline resistance genes Tet (M), tetracycline resistance genes Tet (L), vancomycin resistance genes vanA, and vancomycin resistance genes vanB. Background Art

[0002] Staphylococcus saprophyticus is a Gram-positive bacterium and one of the main pathogens of urinary tract infection. It is one of the common pathogens in uncomplicated urinary tract infection (UTI) in young women. Its clinical features of urinary tract infection have many similarities with those caused by Escherichia coli, but it differs in pathogenesis, seasonal changes and geographical distribution. The incidence of saprophytic Staphylococcus in urinary tract infection is second only to Escherichia coli, especially in women aged 16 to 25 years, where it causes an infection rate of up to 42%. In addition, Staphylococcus aureus can also cause complications such as acute pyelonephritis, urethritis, epididymitis and prostatitis.

[0003] Enterococcus faecalis is the most likely to cause urinary tract infection clinically. This bacteria is very stubborn and difficult to treat. Enterococcus faecalis is resistant to many antibiotics. The antibiotics that can resist this bacteria mainly include penicillin, ampicillin, piperacillin, etc. If the patient has serious complications, they can be used in combination. Causes of urinary tract infection: The vast majority of urinary tract infections are caused by a certain type of bacterial infection, of which more than 90% of patients are caused by Escherichia coli. Clinically, urinary tract infections caused by Enterococcus faecalis are a very common type of infection. Urinary tract infections caused by Enterococcus faecalis are mainly upper and lower urinary tract infections, and a small number of patients present with prostatitis or perinephric abscesses. Sepsis, abdominal infection, pelvic infection, burns, etc. will greatly increase the possibility of Enterococcus faecalis invading the body.

[0004] The main source of Enterococcus faecium may be related to retrograde infection, blood infection, or lymphatic infection. Because the urethra and anus are close, it is possible that bacteria enter the urinary tract through the urethra, resulting in Enterococcus faecium infection; it may also be because there is a fistula between the intestine and the urinary system, and Enterococcus faecium enters the urinary system through the fistula, resulting in Enterococcus faecium infection.

[0005] The gold standard for the diagnosis of clinical urinary tract infection in my country is the culture and identification of clean midstream urine bacteria and drug sensitivity analysis. Given that the culture and identification process is time-consuming overall, clinicians usually use empirical antimicrobial treatment before obtaining the results of drug sensitivity tests. Due to the widespread use of antibiotics in clinical practice, such as aminoglycosides, penicillins, cephalosporins, carbapenems, etc., the distribution and changes of pathogenic microorganisms of urinary tract infections have induced the emergence of drug resistance. Therefore, quickly and accurately obtaining the types of pathogenic microorganisms and drug resistance results can help clinicians quickly select treatment options, improve efficacy and relieve patients' suffering.

[0006] At present, the commonly used detection methods for nucleic acid and drug resistance genes of pathogenic microorganisms of urinary tract infection are mainly fully automatic microbial identification and drug sensitivity identification instruments or nucleic acid detection technology. Automatic identification instruments can detect a wide range of strains and are easy to operate, but they need to isolate and culture pure single colonies of pathogenic microorganisms before they can be tested on the machine, which makes it difficult for the laboratory to quickly detect pathogenic microorganisms and clarify drug resistance. Nucleic acid detection technology is mainly divided into high-throughput sequencing technology and fluorescent PCR method.

[0007] In summary, in order to quickly and comprehensively detect the pathogenic microorganisms and their drug resistance in the patient's urinary system and better guide the rational use of drugs in clinical practice, it is urgent to develop a new technology with wide coverage. The fluorescent PCR method is the most mature and highest-occupancy detection method in the current nucleic acid detection technology market. Due to its high sensitivity, good specificity and controllable cost, it has been widely used in the field of pathogenic microorganism detection. Summary of the invention

[0008] In view of the above-mentioned shortcomings, in the first aspect, the present invention provides a composition for detecting multiple urinary tract infection pathogens and drug resistance genes, wherein the composition comprises at least four groups of primers and probes for detecting the following:

[0009] Upstream and downstream primers and probes for detecting Staphylococcus aureus as shown in SEQ ID NOs: 1 to 3;

[0010] Upstream and downstream primers and probes for detecting Enterococcus faecium as shown in SEQ ID NOs: 4 to 6;

[0011] Upstream and downstream primers and probes for detecting Enterococcus faecalis as shown in SEQ ID NOs: 7 to 9;

[0012] Upstream and downstream primers and probes for detecting the vancomycin resistance gene vanA as shown in SEQ ID NOs: 10 to 12;

[0013] Upstream and downstream primers and probes for detecting the vancomycin resistance gene vanB as shown in SEQ ID NOs: 13 to 15;

[0014] Upstream and downstream primers and probes for detecting tetracycline resistance gene Tet (M) as shown in SEQ ID NOs: 16 to 18;

[0015] The upstream and downstream primers and probes for detecting the tetracycline resistance gene Tet (L) are shown in SEQ ID NOs: 19-21.

[0016] 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 primers, downstream primers and probes that match each other for detecting a target.

[0017] The composition of the present invention can be arbitrarily combined into any combination of detection corresponding to 3 pathogen targets and 4 drug resistance gene 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.

[0018] For example, it may include any two groups of primers and probes for detecting the three groups of pathogens and any three groups of primers and probes for detecting the four groups of drug-resistant genes; it may include any two groups of primers and probes for detecting the three groups of pathogens and any two groups of primers and probes for detecting the four groups of drug-resistant genes; it may include any two groups of primers and probes for detecting the three groups of pathogens and any one group of primers and probes for detecting the four groups of drug-resistant genes; it may include any one group of primers and probes for detecting the three groups of pathogens and any three groups of primers and probes for detecting the four groups of drug-resistant genes, and it may also include any one group of primers and probes for detecting the three groups of pathogens and any two groups of primers and probes for detecting the four groups of drug-resistant genes; it may include any one group of primers and probes for detecting the three groups of pathogens and any one group of primers and probes for detecting the four groups of drug-resistant genes.

[0019] The above-mentioned joint inspection composition provided by the present invention uses a multiplex fluorescence PCR analysis method to detect different pathogens and drug-resistant genes by detecting targets on different pathogens and drug-resistant genes, thereby simultaneously realizing the detection and identification of at least one drug-resistant gene in Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium, as well as tetracycline resistance gene Tet (M), tetracycline resistance gene Tet (L), vancomycin resistance gene vanA, and vancomycin resistance gene vanB in a single-tube reaction system. The composition of the present invention has a higher sensitivity of detection, reaching 200 copies / mL. The present invention can accurately detect pathogens and drug-resistant genes, and can also analyze the mixed infection of bacteria, so as to achieve the effect of precise medication, reduce the use of antibiotics, and save costs. Provide a more sufficient basis for rapid diagnosis for clinicians, and accelerate the implementation of treatment measures for patients.

[0020] Furthermore, the composition comprises:

[0021] Upstream and downstream primers and probes for detecting Staphylococcus aureus as shown in SEQ ID NOs: 1 to 3;

[0022] The upstream and downstream primers and probes for detecting Enterococcus faecium as shown in SEQ ID NOs: 4 to 6; the upstream and downstream primers and probes for detecting Enterococcus faecalis as shown in SEQ ID NOs: 7 to 9;

[0023] The upstream and downstream primers and probes for detecting Enterococcus faecalis as shown in SEQ ID NOs: 10 to 12; the upstream and downstream primers and probes for detecting the vancomycin resistance gene vanB as shown in SEQ ID NOs: 13 to 15;

[0024] Upstream and downstream primers and probes for detecting tetracycline resistance gene Tet (M) as shown in SEQ ID NOs: 16 to 18;

[0025] The upstream and downstream primers and probes for detecting the tetracycline resistance gene Tet (L) are shown in SEQ ID NOs: 19-21.

[0026] The composition of the above-mentioned joint inspection provided by the present invention, by using a multiplex fluorescence PCR analysis method, by detecting targets on different pathogens and drug-resistant genes, different pathogens and drug-resistant genes are detected, thereby simultaneously realizing the detection and identification of 3 pathogens in Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium and 4 drug-resistant genes of tetracycline resistance gene Tet (M), tetracycline resistance gene Tet (L), vancomycin resistance gene vanA and vancomycin resistance gene vanB in a single tube reaction system. The composition of the present invention has a higher sensitivity of detection, reaching 200 copies / mL. The present invention can accurately detect pathogens and drug-resistant genes, and can also analyze the mixed infection of bacteria, so as to achieve the effect of accurate medication, reduce the use of antibiotics, and save costs. Provide a more sufficient basis for rapid diagnosis for clinicians, and accelerate the implementation of treatment measures for patients.

[0027] Furthermore, in the composition, the fluorescent groups of the probes are different from each other and do not interfere with each other.

[0028] In the present invention, "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, ATTO 700, Quasar 705, FAM, VIC, 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.

[0029] In a specific embodiment, the fluorescent reporter group of the probe for detecting Staphylococcus aureus as shown in SEQ ID NO: 3 is FAM; the fluorescent reporter group of the probe for detecting Enterococcus faecium as shown in SEQ ID NO: 6 is HEX; the fluorescent reporter group of the probe for detecting Enterococcus faecalis as shown in SEQ ID NO: 9 is ROX; the fluorescent reporter group of the probe for detecting the vancomycin resistance gene vanA as shown in SEQ ID NO: 12 is CY5; the fluorescent reporter group of the probe for detecting the vancomycin resistance gene vanB as shown in SEQ ID NO: 15 is Quasar 705; the fluorescent reporter group of the probe for detecting the tetracycline resistance gene Tet (M) as shown in SEQ ID NO: 18 is ATTO 425; the fluorescent reporter group of the probe for detecting the tetracycline resistance gene Tet (L) as shown in SEQ ID NO: 21 is ATTO 700.

[0030] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1, BHQ2, or MGB.

[0031] In a specific embodiment, the 3' end of the probe is BHQ1.

[0032] In a specific embodiment, the 3' end of the probe is BHQ2.

[0033] Furthermore, the composition exists in a mixed form.

[0034] Furthermore, the dosage of a single primer in the composition is 0.05 to 5 uM; the dosage of a single probe in the composition is 0.05 to 5 uM.

[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] In a second aspect, the present invention also provides the use of the above-mentioned composition in the preparation of a kit for detecting multiple urinary tract infection pathogens and drug-resistant genes, wherein the pathogens include at least one of Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium; and the drug-resistant genes include at least one of tetracycline resistance gene Tet (M), tetracycline resistance gene Tet (L), vancomycin resistance gene vanA, and vancomycin resistance gene vanB.

[0038] Furthermore, the kit is a fluorescent PCR detection kit.

[0039] In a third aspect, the present invention also provides a kit for detecting a variety of urinary tract infection pathogens and drug-resistant genes, the kit comprising the above-mentioned composition.

[0040] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTPs (U), UNG enzyme, DNA polymerase, PCR buffer and Mg 2+ At least one of .

[0041] Furthermore, the PCR buffer further comprises Tris-HCl, KCl and MgCl 2 Generally, the total volume in a single PCR reaction tube is 20 μL to 100 μL.

[0042] In a specific embodiment, the DNA polymerase is H-Taq enzyme.

[0043] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0044] In a specific embodiment, the negative quality control is at least one of DEPC water and physiological saline. The positive quality control is at least one of the fragment plasmid or fragment DNA or fragment RNA of the specific gene target sequence of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, tetracycline resistance gene Tet (M), tetracycline resistance gene Tet (L), vancomycin resistance gene vanA and vancomycin resistance gene vanB.

[0045] 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.

[0046] In a fourth aspect, the present invention also provides a use of a composition for preparing a detection reagent for multiple urinary tract infection pathogens and drug resistance genes, wherein the detection comprises the following steps:

[0047] S1. Extract or release nucleic acid from the sample to be tested;

[0048] S2. Performing fluorescent quantitative PCR analysis on the nucleic acid obtained in step S1 using the composition described in any one of claims 1 to 5 or the kit described in any one of claims 8 to 9; S3. Obtaining and analyzing the results.

[0049] Furthermore, the reaction conditions of the fluorescent quantitative PCR are: UNG enzyme reaction, 50°C, 2 minutes, 1 cycle; Taq enzyme activation, 95°C, 4 minutes, 1 cycle; denaturation, 95°C, 30 seconds, annealing, extension and fluorescence collection, 60°C, 15 seconds, 45 cycles. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1This is a graph showing the test results of the composition of the present invention;

[0051] Figures 2 to 8 The sensitivity test results of the composition of the present invention are (respectively Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, vanA, vanB, Tet (M), Tet (L);

[0052] Figure 9-15 The precision test results of the composition of the present invention are (respectively Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, vanA, vanB, Tet (M), Tet (L);

[0053] Fig.16 is the specific detection result of the composition of the present invention;

[0054] Fig.17 The test results of the six-joint test of the composition of the present invention;

[0055] Fig.18 The test results of the five-combination test of the composition of the present invention;

[0056] Fig.19 The test results of the quadruple test of the composition of the present invention;

[0057] Fig. 20 The results of single testing of Staphylococcus aureus by the composition of Comparative Example 1 of the present invention;

[0058] Fig.21 The results of a single test of Staphylococcus aureus by the composition of the present invention;

[0059] Fig. 22 The composition of comparative example 1 of the present invention was used to treat Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium. 、 The results of joint testing of tetracycline resistance gene Tet(M), tetracycline resistance gene Tet(L), vancomycin resistance gene vanA, and vancomycin resistance gene vanB. DETAILED DESCRIPTION

[0060] To make the present invention easier to understand, the present invention is further described below in conjunction with specific examples. It should be understood that these examples 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 raw materials and reagents involved in this article can be purchased from the market or prepared by known methods.

[0061] Example 1: Primers and probes used in the present invention

[0062] The primers and probes used in the present invention are shown in Table 1:

[0063] Table 1:

[0064]

[0065]

[0066] Among them, the fluorescent reporter group of the probe for detecting Staphylococcus aureus is FAM; the fluorescent reporter group of the probe for detecting Enterococcus faecium is HEX; the fluorescent reporter group of the probe for detecting Enterococcus faecalis is ROX; the fluorescent reporter group of the probe for detecting vancomycin resistance gene vanA is CY5; the fluorescent reporter group of the probe for detecting vancomycin resistance gene vanB is Quasar705; the fluorescent reporter group of the probe for detecting tetracycline resistance gene Tet (M) is ATTO 425; the fluorescent reporter group of the probe for detecting tetracycline resistance gene Tet (L) is ATTO 700.

[0067] Example 2: Method for detecting multiple urinary tract infection pathogens and drug resistance genes

[0068] (1) Reagent preparation:

[0069] Take the corresponding amount of PCR buffer and enzyme mixture (PCR buffer 40 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.

[0070] PCR buffer consists of Tris-HCl, MgCl 2 , KCl, Triton X-100 and other buffer systems.

[0071] 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.

[0072] (2) Sample processing and sample addition:

[0073] 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.

[0074] 10uL of the above-treated samples, negative controls and positive controls were pipetted into reaction tubes respectively, and 40uL of PCR mixture was added to each reaction tube and the tube cap was covered.

[0075] The reaction systems of the above-mentioned original reaction tubes are shown in Table 2 below:

[0076] Table 2:

[0077] Components The volume of each reaction PCR buffer 34.3 dNTPs(U)(100mM) 0.5 <![CDATA[1mol / LMgcl 2 ]]> 0.2 Primer SEQ ID NO: 1 (50 pmol / μL) 0.2 Primer SEQ ID NO: 2 (50 pmol / μL) 0.2 Probe SEQ ID NO: 3 (50 pmol / μL) 0.1 Primer SEQ ID NO: 4 (50 pmol / μL) 0.2 Primer SEQ ID NO: 5 (50 pmol / μL) 0.2 Probe SEQ ID NO: 6 (50 pmol / μL) 0.1 Primer SEQ ID NO:7 (50 pmol / μL) 0.2 Primer SEQ ID NO: 8 (50 pmol / μL) 0.2 Probe SEQ ID NO: 9 (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 Probe SEQ ID NO: 12 (50 pmol / μL) 0.1 Primer SEQ ID NO: 13 (50 pmol / μL) 0.2 Primer SEQ ID NO: 14 (50 pmol / μL) 0.2 Probe SEQ ID NO: 15 (50 pmol / μL) 0.1 Primer SEQ ID NO: 16 (50 pmol / μL) 0.2 Primer SEQ ID NO: 17 (50 pmol / μL) 0.2 Probe SEQ ID NO: 18 (50 pmol / μL) 0.1 Primer SEQ ID NO: 19 (50 pmol / μL) 0.2 Primer SEQ ID NO: 20 (50 pmol / μL) 0.2 Probe SEQ ID NO: 21 (50 pmol / μL) 0.1

[0078] (3) PCR amplification:

[0079] 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:

[0080] Table 3:

[0081]

[0082] (4) Interpretation of test results

[0083] If the amplification curve is not present and the Ct value is ≤40, it is considered positive; if there is no amplification curve (No Ct) or the Ct value is greater than 40 and CY5≤40 for ATTO 425, FAM, HEX, ROX, CY5, Quasar 705 and ATTO 700 channels, it is considered negative; if there is no amplification curve (No Ct) or the Ct value is greater than 40 and CY5 is greater than 40 for ATTO 425, FAM, HEX, ROX and Quasar 705 channels, the result is unreliable and needs to be retested; as shown in Table 4:

[0084] Table 4:

[0085]

[0086] Example 3: Test results of the test samples of the composition of the present invention

[0087] 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 of various pathogen-positive (Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium) and drug-resistant genes (tetracycline resistance gene Tet (M), tetracycline resistance gene Tet (L), vancomycin resistance gene vanA, vancomycin resistance gene vanB). The detection results are as follows: Figure 1 As shown. Figure 1 It can be seen that the composition of the present application can simultaneously detect tetracycline resistance gene Tet (M), tetracycline resistance gene Tet (L), vancomycin resistance gene vanA, and vancomycin resistance gene vanB.

[0088] Example 4: Sensitivity of the composition of the present invention

[0089] The positive samples were diluted with negative samples to a concentration of 200 copies / mL, and the test was repeated 20 times to verify the sensitivity of the composition and kit of the present invention. Figures 2 to 8 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.

[0090] Table 5:

[0091]

[0092] Example 5: Precision of the composition of the present invention

[0093] The positive samples were diluted to a medium concentration (400 copies / mL) for precision determination, and each sample was measured 10 times according to the method of Example 2. The results are shown in Figure 9-15 As shown, the detection rate of each target is 100%, and the coefficient of variation (CV) of the Ct value is less than 5%. It can be considered that this kit has good detection precision both within and between batches.

[0094] Example 6. Specificity of the composition of the present invention

[0095] 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 (Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Candida albicans, Mycoplasma hominis, Trichomonas vaginalis, Escherichia coli, Pseudomonas aeruginosa) that have homology in nucleic acid sequence and are likely to cause the same or similar clinical symptoms. The results are shown in the following table. Fig.16 As shown, from Fig.16 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.

[0096] Example 7: Anti-interference performance of the composition of the present invention

[0097] Interferors were analyzed according to the method described in Example 2. The experimental results showed that potential PCR inhibitors / interfering substances such as gentamicin (50 μg / mL), dexamethasone hydrochloride (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:

[0098] Table 6:

[0099]

[0100] Example 8: Six-way test of the composition of the present invention

[0101] According to the primers and probes shown in Table 2, Staphylococcus aureus, Enterococcus faecalis, and drug-resistant genes (tetracycline resistance gene Tet (M), tetracycline resistance gene Tet (L), vancomycin resistance gene vanA, and vancomycin resistance gene vanB) can also be detected and distinguished by six joint tests. The test results are as follows: Fig.17 shown.

[0102] Example 9, Five-combination test of the composition of the present invention

[0103] According to the primers and probes shown in Table 2, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, and drug-resistant genes (vancomycin resistance gene vanA, vancomycin resistance gene vanB) can also be tested and differentiated. The test results are as follows: Fig.18 shown.

[0104] Example 10: Quadruple test of the composition of the present invention

[0105] According to the primers and probes shown in Table 2, Enterococcus faecalis, Enterococcus faecium, and drug-resistant genes (vancomycin resistance gene vanA, vancomycin resistance gene vanB) can also be detected and distinguished. The test results are as follows: Fig.19 shown.

[0106] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0107] 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.

[0108] Therefore, the inventors also designed some other primers and probes, such as another pair of Staphylococcus aureus primers and probes (sequence not shown), which were replaced with the primers and probes of Staphylococcus aureus in the kit of the present invention, and the detection systems before and after the replacement were used to test Klebsiella pneumoniae alone, and the results were as follows: Figure 20-21 As shown. Figure 20-21It can be seen that the detection system before and after the replacement has the same effect on the single detection of Klebsiella pneumoniae. The replaced detection system was used to conduct a joint test on three cocci and four drug-resistant specific genes (Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, and tetracycline resistance gene Tet (M), tetracycline resistance gene Tet (L), vancomycin resistance gene vanA, and vancomycin resistance gene vanB). The results are as follows Fig. 22 As shown. Fig. 22 and Figure 1 From the comparison of the amplification curves, it can be seen that when the comparative example composition is used for joint detection, the performance of Staphylococcus aureus is reduced, the Ct value is delayed, and the fluorescence increment decreases. This shows that the combination of primers and probes in the present invention is irreplaceable.

[0109] 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 detecting multiple urinary tract infection pathogens and drug resistance genes, characterized in that: The composition includes at least four sets of primers and probes for detecting: Upstream and downstream primers and probes for detecting Staphylococcus aureus as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Enterococcus faecium as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Enterococcus faecalis as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting the vancomycin resistance gene vanA as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes for detecting the vancomycin resistance gene vanB as shown in SEQ ID NOs: 13 to 15; Upstream and downstream primers and probes for detecting tetracycline resistance gene Tet (M) as shown in SEQ ID NOs: 16 to 18; The upstream and downstream primers and probes for detecting the tetracycline resistance gene Tet (L) are shown in SEQ ID NOs: 19-21.

2. The composition according to claim 1, characterized in that The composition comprises: Upstream and downstream primers and probes for detecting Staphylococcus aureus as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Enterococcus faecium as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Enterococcus faecalis as shown in SEQ ID NOs: 7 to 9; The upstream and downstream primers and probes for detecting Enterococcus faecalis as shown in SEQ ID NOs: 10 to 12; the upstream and downstream primers and probes for detecting the vancomycin resistance gene vanB as shown in SEQ ID NOs: 13 to 15; Upstream and downstream primers and probes for detecting tetracycline resistance gene Tet (M) as shown in SEQ ID NOs: 16 to 18; The upstream and downstream primers and probes for detecting the tetracycline resistance gene Tet (L) are shown in SEQ ID NOs: 19-21.

3. 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.

4. The composition according to claim 3, characterized in that The fluorescent reporter group of the probe for detecting Staphylococcus aureus as shown in SEQ ID NO: 3 is FAM; The fluorescent reporter group of the probe for detecting Enterococcus faecium as shown in SEQ ID NO: 6 is HEX; The fluorescent reporter group of the probe for detecting Enterococcus faecalis as shown in SEQ ID NO: 9 is ROX; The fluorescent reporter group of the probe for detecting the vancomycin resistance gene vanA as shown in SEQ ID NO: 12 is CY5; The fluorescent reporter group of the probe for detecting the vancomycin resistance gene vanB as shown in SEQ ID NO: 15 is Quasar705; The fluorescent reporter group of the probe for detecting the tetracycline resistance gene Tet (M) as shown in SEQ ID NO: 18 is ATTO 425; As shown in SEQ ID NO: 21, the fluorescent reporter group of the probe for detecting the tetracycline resistance gene Tet (L) is ATTO 700.

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

6. Use of the composition according to any one of claims 1 to 5 in preparing a kit for detecting multiple urinary tract infection pathogens and drug resistance genes, characterized in that: The pathogenic bacteria include at least one of Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium; the drug-resistant gene includes at least one of tetracycline resistance gene Tet (M), tetracycline resistance gene Tet (L), vancomycin resistance gene vanA, and vancomycin resistance gene vanB.

7. The use according to claim 6, characterized in that: The kit is a fluorescent PCR detection kit.

8. A kit for detecting multiple urinary tract infection pathogens and drug resistance genes, characterized in that: The kit comprises the composition according to any one of claims 1 to 5.

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 (U), UNG enzyme, DNA polymerase, PCR buffer and Mg 2+ At least one of .

10. Use of a composition for preparing a detection reagent for multiple 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 5 or the kit according to any one of claims 8 to 9; S3. Obtain and analyze the results.

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