Composition and kit for simultaneously detecting five urinary tract infection pathogenic bacteria 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 a kit for detecting serratia marcescens, neisseria meningitidis, providencia stuartii, proteus mirabilis and proteus vulgaris and application of the composition and the kit. Primer probes are designed for conserved sequences of pathogens by adopting a multiplex PCR technology, mutual interference among the primer probes is avoided, five pathogens can be simultaneously detected in one tube of reaction liquid, and time-consuming and labor-consuming operation of a traditional bacterial culture method is changed to a great extent. Meanwhile, the composition is accurate in detection result and has excellent precision, sensitivity and specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro nucleic acid detection in molecular biology, and in particular to the detection of five urinary tract infection pathogens, and more particularly to the joint detection of Serratia marcescens, Neisseria meningitidis, Providencia stuartii, Proteus mirabilis and Proteus vulgaris. Background Art

[0002] Serratia marcescens, also known as Bacillus marcescens, is the smallest bacterium that normally exists in the human body. The urinary tract and respiratory tract are important reservoirs of bacteria, and the gastrointestinal tract of children can also store bacteria. It is an important conditional pathogen of nosocomial infection. When the body's immune function is low, it can cause pneumonia, urinary tract infection, sepsis, and meningitis. It is resistant to multiple antibiotics and can cause pneumonia, sepsis, surgical infection and other infections. Due to the presence of plasmids that carry genes encoding drug resistance, most Serratia marcescens are resistant to multiple antibiotics such as ampicillin and macrolides. In recent years, with the use of immunosuppressants and a large number of broad-spectrum antibiotics, hospital infections caused by Serratia marcescens have continued to increase, and resistance to most antimicrobial drugs has become increasingly serious, bringing difficulties to clinical anti-infection treatment.

[0003] Neisseria meningitidis is a gram-negative aerobic diplococcus belonging to the family Neisseriaceae and is the causative agent of epidemic cerebrospinal meningitis. In addition, Neisseria meningitidis may also cause lung infections, joint infections, respiratory tract infections, genitourinary system infections, rectal infections, eye infections, endocarditis, and pericarditis, but this is rare. More than 90% of cases of meningococcal infection are meningitis or meningococcemia, but urethritis and cervicitis have also been reported. The incidence of meningococcal urethritis has been increasing in men who have sex with men, usually after reproductive contact with oropharyngeal meningococcal carriers.

[0004] Providencia stuartii belongs to the family Enterobacteriaceae and is a Gram-negative bacillus. As a conditional pathogen, it can cause urinary tract infection under certain conditions, especially in elderly male patients with underlying diseases and long-term indwelling catheterization. Anti-infection treatment usually uses third- and fourth-generation cephalosporins or anti-pseudomonal penicillins + enzyme inhibitors. However, Providencia stuartii is more resistant to certain antibiotics, with a high proportion of multidrug-resistant (MDR) strains, and the mortality rate of patients infected with pan-drug-resistant (PDR) strains is significantly higher than that of non-pan-drug-resistant patients. Therefore, the monitoring of Providencia stuartii infection should be strengthened, antimicrobial drugs should be used rationally, and the spread of pan-drug-resistant strains should be avoided.

[0005] Proteus also belongs to the Enterobacteriaceae family, is Gram-negative, widely found in nature, and can also exist in the intestines of humans or animals. It is an important conditional pathogen. Currently, the incidence of Proteus in the urinary system is of greatest concern. Among the bacteria of this genus, infection caused by Proteus mirabilis is the most common. Proteus mirabilis is the second most common pathogen causing urinary tract infection clinically, and can also cause enteritis in infants and young children. The second most common is Proteus vulgaris, which can cause urinary tract infection, respiratory tract infection, peritonitis, gastroenteritis, wound infection, bacteremia, meningitis and many other infections.

[0006] Urinary tract infection is an inflammatory disease caused by the abnormal growth and reproduction of various pathogenic microorganisms in the urinary tract. It is one of the most common infectious diseases. Every year, 130 million to 175 million people suffer from urinary tract infection worldwide, which is the second most common infectious disease after respiratory tract infection. Urinary tract infection often manifests as frequent urination, urgency, dysuria, chills, fever, back pain, bacteriuria, pyuria, etc. Pathogens may include bacteria, fungi, mycoplasma, chlamydia and other categories. At present, the diagnosis of urinary tract infection is mainly based on routine urine examination, urinary system contrast examination and urine culture. Urine bacterial culture is the gold standard for the diagnosis of pathogenic bacteria of urinary tract infection, but the isolation and culture of pathogens is time-consuming and cumbersome. Compared with other detection methods, real-time fluorescence quantitative PCR technology is more suitable for early detection, with higher sensitivity and specificity, and can accurately and quickly detect Serratia marcescens, Neisseria meningitidis, Providencia stuartii, Proteus mirabilis and Proteus vulgaris. Summary of the invention

[0007] In view of this, in a first aspect, the present invention provides a composition for simultaneously detecting five urinary tract infection pathogens, the composition comprising at least four of the following primers and probes for detecting urinary tract infection pathogens:

[0008] The upstream and downstream primers and probes of Serratia marcescens as shown in SEQ ID NOs: 1 to 3;

[0009] Upstream and downstream primers and probes of Neisseria meningitidis as shown in SEQ ID NOs: 4 to 6;

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

[0011] Upstream and downstream primers and probes of Proteus mirabilis as shown in SEQ ID NOs: 10 to 12;

[0012] The upstream and downstream primers and probes of Proteus vulgaris are shown in SEQ ID NOs: 13-15.

[0013] The present invention adopts multiplex PCR technology to design primer probes based on the conservative sequences of each pathogen, and ensures that there is no mutual interference between the primer probes. Five pathogens can be detected simultaneously in one tube of reaction solution, which greatly changes the time-consuming and laborious operation of the traditional bacterial culture method. At the same time, the composition of the present invention has accurate detection results and excellent precision, sensitivity and specificity.

[0014] In some embodiments, the composition of the present invention may include one or more sets of the above primers and probes. In this article, "set" refers to upstream primers, downstream primers and probes that match each other to detect a target.

[0015] Furthermore, the composition of the present invention can be arbitrarily 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 sets corresponding to the targets. These combinations are all included in the present invention.

[0016] For example, it may include all combinations of the above-mentioned 5 groups of primers and probes, it may include any 4 groups of the above-mentioned 5 groups of primers and probes, it may include any 3 groups of the above-mentioned 5 groups of primers and probes, it may include any 2 groups of the above-mentioned 5 groups of primers and probes, and it may include any 1 group of the above-mentioned 5 groups of primers and probes.

[0017] Furthermore, in the composition, the fluorescent groups of the probes are different from each other and do not interfere with each other.

[0018] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, FAM, HEX, ROX, CY5 and Quasar 705 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.

[0019] Furthermore, the 5' end of the probe in the composition of the present invention is labeled with a fluorescent reporter group.

[0020] In a specific embodiment, the fluorescent reporter group of the probe for Serratia marcescens as shown in SEQ ID NO:3 is FAM; the fluorescent reporter group of the probe for Neisseria meningitidis as shown in SEQ ID NO:6 is HEX; the fluorescent reporter group of the probe for Providencia stuartii as shown in SEQ ID NO:9 is ROX; the fluorescent reporter group of the probe for Proteus mirabilis as shown in SEQ ID NO:12 is CY5; and the fluorescent reporter group of the probe for Proteus vulgaris as shown in SEQ ID NO:15 is Quasar 705.

[0021] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1, BHQ2, or MGB.

[0022] In a specific embodiment, the 3' end of the probe is BHQ1.

[0023] In a specific embodiment, the 3' end of the probe is BHQ2.

[0024] Furthermore, the composition exists in a mixed form.

[0025] In a second aspect, the present invention provides a use of the above-mentioned composition in preparing a kit for simultaneously detecting five urinary tract infection pathogens, wherein the five urinary tract infection pathogens are Serratia marcescens, Neisseria meningitidis, Providencia stuartii, Proteus mirabilis and Proteus vulgaris.

[0026] Furthermore, the kit is a fluorescent PCR detection kit.

[0027] In a third aspect, the present invention further provides a kit for simultaneously detecting five urinary tract infection pathogens, wherein the kit comprises the above-mentioned composition of the present invention.

[0028] In a specific embodiment, in each reaction system, the concentration of each primer is 50 pmol / μL, and the concentration of each probe is 50 pmol / μL.

[0029] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0030] In a specific embodiment, the negative control is at least one of normal saline and DEPC H2O.

[0031] In a specific embodiment, the positive control is at least one of a fragmented plasmid or a fragmented DNA of Serratia marcescens, Neisseria meningitidis, Providencia stuartii, Proteus mirabilis, and Proteus vulgaris.

[0032] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, a PCR buffer, UNG enzyme, DNA polymerase, dNTP (U), Mg 2+ At least one of .

[0033] In some specific embodiments, the DNA polymerase is H-Taq enzyme.

[0034] In some specific embodiments, the kit of the present invention includes PCR buffer, UNG enzyme, H-Taq enzyme, dNTP (U), Mg 2+ .

[0035] Common PCR buffers are composed of buffer systems such as Tris-HCl, KCl, and NaCl2.

[0036] In a fourth aspect, the present invention provides a use of a composition for preparing a detection kit for simultaneously detecting five urinary tract infection pathogens, comprising the following steps:

[0037] S1. Extract or release nucleic acid from the sample to be tested;

[0038] S2. Performing fluorescent quantitative PCR analysis on the nucleic acid obtained in step S1 using the composition of the present invention or the kit of the present invention;

[0039] S3. Obtain and analyze the results.

[0040] In the present invention, the sample used for detection is urine, but is not limited thereto.

[0041] Furthermore, the reaction conditions of the fluorescent quantitative PCR analysis are:

[0042] UNG decontamination, 50°C, 5 minutes, 1 cycle; pre-denaturation, 95°C, 5 minutes, 1 cycle; denaturation, 95°C, 10 seconds, annealing, extension, and fluorescence collection, 58°C, 25 seconds, 45 cycles; instrument cooling (optional), 25°C, 10 seconds, 1 cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is the test result of the composition of the present invention;

[0044] Figure 2 is the amplification effect of the composition of the present invention on the detection limit concentration of Serratia marcescens;

[0045] Figure 3 is the amplification effect of the composition of the present invention on the detection limit concentration of Neisseria meningitidis;

[0046] Figure 4 is the amplification effect of the composition of the present invention on the detection limit concentration of Providencia stuartii;

[0047] Figure 5 is the amplification effect of the composition of the present invention on the detection limit concentration of Proteus mirabilis;

[0048] Figure 6 is the amplification effect of the composition of the present invention on the detection limit concentration of Proteus vulgaris;

[0049] Figure 7 It is the specificity detection result of the composition of the present invention;

[0050] Figure 8 This is the precision test result of the composition of the present invention on Serratia marcescens;

[0051] Fig. 9 The precision test result of the composition of the present invention on Neisseria meningitidis;

[0052] Fig.10 The precision test result of the composition of the present invention on Providencia stuartii;

[0053] Fig.11 The precision test result of the composition of the present invention on Proteus mirabilis;

[0054] Fig.12 This is the precision test result of the composition of the present invention on Proteus vulgaris;

[0055] Fig.13 The single test results of the primers and probes of Serratia marcescens of the present invention and the primers and probes of Serratia marcescens of Comparative Example 1 are shown;

[0056] Fig.14 The results of the five-joint test of the composition of the present invention, the primers and probes for Serratia marcescens in comparative example 1, and the primers and probes for the remaining four targets of the composition of the present invention are shown. DETAILED DESCRIPTION

[0057] To make the present invention easier to understand, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by professional and technical personnel in the field; unless otherwise specified, the instruments and reagents involved in this article are conventional instruments and reagents in the field.

[0058] Example 1: Primers and probes used in the present invention

[0059] Table 1:

[0060]

[0061] Among them, the fluorescent reporter group of the probe SM-P1 of Serratia marcescens is FAM; the fluorescent reporter group of the probe NM-P2 of Neisseria meningitidis is HEX; the fluorescent reporter group of the probe PS-P1 of Providencia stuartii is ROX; the fluorescent reporter group of the probe PM-P2 of Proteus mirabilis is CY5; and the fluorescent reporter group of the probe PV-P2 of Proteus vulgaris is Quasar705.

[0062] Example 2: Method for simultaneously detecting five urinary tract infection pathogens

[0063] (1) Establishment of amplification system

[0064] The present invention designs a tube of reaction solution based on polymerase chain reaction (PCR). The PCR reaction system comprises at least the following components: PCR buffer, UNG enzyme, DNA polymerase, dNTPs (U), primers and probes, and metal cations required for catalyzing DNA polymerase reaction. Common PCR buffers are composed of buffer systems such as Tris-HCl, KCl, and NaCl2.

[0065] The real-time fluorescent PCR reaction system of the present invention is configured according to the following Table 2:

[0066] Table 2:

[0067]

[0068]

[0069] (2) Reagent preparation

[0070] According to the number of samples to be tested, positive controls, and negative controls, take the corresponding amount of PCR reaction solution and enzyme in proportion, mix them thoroughly to form a PCR system, and centrifuge at 2000 rpm for 10 seconds before use.

[0071] (3) Sample processing and sample addition

[0072] The sample to be tested in the embodiment of the present invention is urine. Take 300uL of the sample to be tested, the negative control, and the positive control into a 1.5mL centrifuge tube, and use the commercially available nucleic acid extraction or purification reagent of Shengxiang Biotechnology Co., Ltd. to extract nucleic acid according to its instructions; 10uL of the above-treated sample, negative control, and positive control are respectively added to the corresponding 0.2mL PCR reaction tubes, 40uL of PCR mixed solution is added to each tube, and the tube cap is covered.

[0073] (4) PCR amplification

[0074] PCR amplification was performed on a Hongshi SLAN-96PCR instrument according to the temperature and time settings shown in Table 3.

[0075] Table 3:

[0076]

[0077]

[0078] (4) Result analysis and judgment

[0079] If the FAM, HEX, CY5, ROX, Quasar 705 channels have obvious S-shaped amplification curves and the CT value is ≤38, it is judged as positive; if the FAM, HEX, CY5, ROX, Quasar 705 channels have no amplification curve (No CT) or the CT value is greater than 38, it is judged as negative. The details are shown in Table 4:

[0080] Table 4:

[0081]

[0082] Invalid results: No typical S-shaped amplification curve was detected for Serratia marcescens (FAM), Neisseria meningitidis (HEX), Providencia stuartii (ROX), Proteus mirabilis (CY5) and Proteus vulgaris (Quasar705), indicating that there are interfering substances in the specimen. If the result is invalid again after retesting, fresh samples should be collected for retesting.

[0083] Example 3: Test results of the test samples of the composition of the present invention

[0084] The primers and probes shown in Example 1 were used according to the method of Example 2 to test the samples (Serratia marcescens, Neisseria meningitidis, Providencia stuartii, Proteus mirabilis and Proteus vulgaris were all positive) on a Hongshi SLAN96 real-time fluorescence quantitative PCR instrument to verify the composition of the present invention. The test results are shown in FIG. Figure 1 As shown, from Figure 1 It can be seen that the composition of the present invention can well detect and distinguish the gene fragments of Serratia marcescens, Neisseria meningitidis, Providencia stuartii, Proteus mirabilis and Proteus vulgaris.

[0085] Example 4: Sensitivity of the composition of the present invention

[0086] Using the composition of the present invention, the positive samples of Serratia marcescens, Neisseria meningitidis, Providencia stuartii, Proteus mirabilis and Proteus vulgaris were diluted to a concentration of 500 copies / mL for LOD (sensitivity) detection. The results are shown in Table 5. The corresponding amplification effects are as follows Figures 2 to 6 As shown by Figures 2 to 6 It can be seen that the test results show that the composition can still accurately detect samples as low as 500 copies / mL in each channel, indicating that the sensitivity of the composition of the present invention is 500 copies / mL.

[0087] Table 5:

[0088]

[0089] Example 5. Specificity of the composition of the present invention

[0090] 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 (Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Mobiluncus, Pseudomonas aeruginosa, Candida albicans, Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis) 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. Figure 7 As shown, from Figure 7 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.

[0091] Example 6: Precision of the composition of the present invention

[0092] The composition of the present invention was used to perform precision testing on positive samples of Serratia marcescens, Neisseria meningitidis, Providencia stuartii, Proteus mirabilis and Proteus vulgaris, and each sample was tested 10 times. The results are shown in FIG. Figure 8-12 As shown, the detection rates of these samples were all 100%, and the coefficient of variation (CV) of the Ct value was less than 5%. It can be considered that this kit has good detection precision both within and between batches.

[0093] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0094] 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.

[0095] In the present invention, the combination of primers and probes is irreplaceable and non-replaceable. Another primer and probe for Serratia marcescens used in the comparative example composition (see Table 6, the upstream and downstream primers and probes are shown in SEQ ID NOs: 16 to 18 in sequence) and the primer and probe for Serratia marcescens used in the composition of the present invention (the upstream and downstream primers and probes are shown in SEQ ID NOs: 1 to 3 in sequence) were tested separately, and the results are shown in FIG. Fig.13 As shown by Fig.13 It can be seen that the detection effect is very good and comparable in the single test system. The primers and probes for Serratia marcescens of Comparative Example 1 were combined with the primers and probes for the other four targets of the composition of the present invention, and the samples to be tested in Example 3 (Serratia marcescens, Neisseria meningitidis, Providencia stuartii, Proteus mirabilis and Proteus vulgaris were all positive) were subjected to a five-joint test, and the results are as follows: Fig.14 As shown by Fig.14 It can be seen that the primers and probes for Serratia marcescens set in this comparative example are affected in the five-in-one detection system, and there is a significant reduction in detection performance, and even affects the detection of other targets, which further illustrates the superiority of the composition of the present invention.

[0096] Table 6:

[0097]

[0098] In summary, the present invention uses multiplex PCR technology to design primer probes for the conserved sequences of each pathogen, and ensures that there is no mutual interference between the primer probes. Five pathogens can be detected simultaneously in one tube of reaction solution, which greatly changes the time-consuming and laborious operation of the traditional bacterial culture method. At the same time, the composition of the present invention has accurate detection results and excellent precision, sensitivity and specificity.

[0099] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A composition for simultaneously detecting five urinary tract infection pathogens, characterized in that: The composition includes at least 4 of the following primers and probes for detecting urinary tract infection pathogens: The upstream and downstream primers and probes of Serratia marcescens as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes of Neisseria meningitidis as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes of Providencia stuartii as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes of Proteus mirabilis as shown in SEQ ID NOs: 10 to 12; The upstream and downstream primers and probes of Proteus vulgaris are shown in SEQ ID NOs: 13-15.

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

3. The composition according to claim 2, characterized in that The fluorescent reporter group of the probe for Serratia marcescens as shown in SEQ ID NO: 3 is FAM; The fluorescent reporter group of the probe for Neisseria meningitidis as shown in SEQ ID NO: 6 is HEX; The fluorescent reporter group of the probe of Providencia stuartii as shown in SEQ ID NO:9 is ROX; The fluorescent reporter group of the probe of Proteus mirabilis as shown in SEQ ID NO: 12 is CY5; The fluorescent reporter group of the probe for Proteus vulgaris as shown in SEQ ID NO:15 is Quasar 705.

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

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

6. The use according to claim 5, characterized in that The five urinary tract infection pathogens are Serratia marcescens, Neisseria meningitidis, Providencia stuartii, Proteus mirabilis, and Proteus vulgaris.

7. The use according to claim 5, characterized in that The kit is a fluorescent PCR detection kit.

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

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

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

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