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

Abstract

The invention belongs to the technical field of in-vitro nucleic acid detection of molecular biology, and particularly relates to a composition and a method for detecting and distinguishing mycoplasma hominis, ureaplasma urealyticum, actinophora skinneri, fluoroquinolones drug-resistant genes GyrA and tetracycline drug-resistant genes tetB. The joint detection composition provided by the invention utilizes a PCR (Polymerase Chain Reaction) detection technology to detect and distinguish mycoplasma hominis, ureaplasma urealyticum, actinophora skinneri, fluoroquinolones drug-resistant genes GyrA and tetracycline drug-resistant genes tetB; three common urinary tract infection pathogenic bacteria and two drug-resistant gene segments can be rapidly detected in one tube of reaction liquid. The method has the advantages of short time, high sensitivity, good specificity and the like, and can be used for accurately and quickly detecting mycoplasma hominis, ureaplasma urealyticum, actinobacillus sterneri, fluoroquinolone drug-resistant genes GyrA and tetracycline drug-resistant genes tetB.

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 three common urinary tract infection pathogens and two drug resistance genes; more specifically, to the detection of Mycoplasma hominis, Ureaplasma urealyticum, Actinomyces steadiensis, fluoroquinolone resistance gene GyrA, and tetracycline resistance gene tetB. Background Art

[0002] Mycoplasma hominis (MH) is a type of mycoplasma that exists in the urinary system and genitals and can cause urinary system infections and genital inflammation. MH is associated with a variety of urogenital tract infections such as non-gonococcal urethritis, female cervicitis, adnexitis and infertility. The infection site in adult males is in the urethral mucosa, and the infection site in females is in the cervix. Long-term or repeated chlamydia and mycoplasma infections can destroy the endometrial layer in the cervix and fallopian tubes, causing infertility. In newborns, it mainly causes conjunctivitis and pneumonia.

[0003] Ureaplasma urealyticum is the smallest prokaryotic organism between bacteria and viruses. It mainly lives in the human urogenital tract, of which Ureaplasma Urealyticum (UU) is a type of Ureaplasma urealyticum, which is one of the important pathogens of sexually transmitted diseases. Ureaplasma urealyticum is highly contagious, and the most common routes of transmission include oral, vaginal and anal contact. Ureaplasma urealyticum can not only cause genital infections such as vagina and cervix, but is also one of the common parasites in the human urogenital system, and is closely related to miscarriage, infertility and intrauterine growth retardation of the fetus. Ureaplasma urealyticum infection usually has no symptoms, so most people do not realize that they have been infected. If not treated in time, Ureaplasma urealyticum infection may cause infertility, premature birth or stillbirth, chorioamnionitis, urethritis, prostatitis, epididymitis, meningitis and pneumonia. It can also spread to other parts of the body, causing reactive arthritis and long-term damage to joints, nerves and muscles.

[0004] Actinocotina schaalii is a Gram-positive, facultative anaerobic bacterium belonging to the phylum Actinobacteria. It grows slowly under aerobic conditions and usually forms small white colonies on blood agar plates. This bacterium is widely distributed in the natural environment, especially in soil. Actinocotina schaalii is closely related to human health. It is a common pathogen of urinary tract infections, especially in the elderly and children. In addition, it may also cause invasive diseases such as bacteremia, endocarditis, discitis, and hidradenitis.

[0005] Fluoroquinolone resistance genes refer to genes that confer resistance to fluoroquinolone antibiotics. Fluoroquinolone antibiotics are synthetic antibacterial drugs that block DNA replication and RNA synthesis by inhibiting bacterial topoisomerases (such as DNA gyrase and topoisomerase IV), thereby exerting a bactericidal effect. Fluoroquinolone drugs target bacterial DNA gyrase and topoisomerase IV. Mutations in the genes encoding these enzymes (such as *gyrA*, *gyrB*, *parC*, and *parE*) can reduce the affinity of the drug to the target protein, leading to drug resistance. Certain resistance genes such as *qnr*, *aac(6')-Ib-cr*, and *oqxA / B* can be spread between bacteria through plasmids, leading to the spread of drug resistance. Reduced permeability of the cell wall or membrane reduces the entry of drugs, which is also one of the mechanisms of drug resistance. The emergence and spread of drug resistance poses a serious threat to public health and needs to be controlled through the rational use of antibiotics and the development of new antibacterial strategies.

[0006] Tetracycline resistance is usually due to the acquisition of new genes that encode proteins that regulate the energy-dependent efflux of tetracycline or protect the bacterial ribosome from the action of tetracycline. Many of these genes are associated with mobile plasmids or transposons and can be distinguished from one another using molecular methods, including DNA-DNA hybridization and DNA sequencing using oligonucleotide probes. A limited number of bacteria acquire resistance through mutations that alter the permeability of outer membrane porins and / or outer membrane lipopolysaccharide, change the regulation of the innate efflux system, or alter the 16S rRNA. Summary of the invention

[0007] In view of the above-mentioned shortcomings, the present invention provides a composition for simultaneously detecting three common urinary tract infection pathogens and two drug resistance genes, wherein the composition comprises at least four groups of primers and probes for detecting the following:

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

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

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

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

[0012] The upstream and downstream primers and probes for detecting the tetracycline resistance gene tetB are shown in SEQ ID NOs: 13-15.

[0013] Further, a composition for simultaneously detecting three common urinary tract infection pathogens and two drug resistance genes, the composition comprises five groups of primers and probes for detecting the following:

[0014] Upstream and downstream primers and probes for detecting Mycoplasma hominis as shown in SEQ ID NOs: 1 to 3;

[0015] Upstream and downstream primers and probes for detecting Ureaplasma urealyticum as shown in SEQ ID NOs: 4 to 6;

[0016] Upstream and downstream primers and probes for detecting Actinocoryneformis as shown in SEQ ID NOs: 7 to 9;

[0017] Upstream and downstream primers and probes for detecting the fluoroquinolone resistance gene GyrA as shown in SEQ ID NOs: 10 to 12;

[0018] The upstream and downstream primers and probes for detecting the tetracycline resistance gene tetB are shown in SEQ ID NOs: 13-15.

[0019] The above-mentioned joint detection composition provided by the present invention can realize the rapid detection of three common urinary tract infection pathogens and two drug-resistant gene fragments in one tube of reaction liquid by using PCR detection technology. The present invention has the advantages of short time, high sensitivity and good specificity, and can accurately and quickly detect Mycoplasma hominis, Ureaplasma urealyticum, Actinomycetemcomitans, fluoroquinolone resistance gene GyrA and tetracycline resistance gene tetB.

[0020] Furthermore, the 5' end of the probe in the composition is labeled with a fluorescent group.

[0021] Furthermore, in the composition, the fluorescent groups of the probes are different from each other and do not interfere with each other.

[0022] 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, 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.

[0023] 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 primers, downstream primers and probes that match each other to detect a target.

[0024] 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.

[0025] 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.

[0026] In a specific embodiment, the fluorescent group of the probe shown in SEQ ID NO: 3 is FAM; the fluorescent group of the probe shown in SEQ ID NO: 6 is HXE; the fluorescent group of the probe shown in SEQ ID NO: 9 is ROX; the fluorescent group of the probe shown in SEQ ID NO: 12 is CY5; the fluorescent group of the probe shown in SEQ ID NO: 15 is Quasar705.

[0027] Furthermore, the 3' end of the probe also has a quenching group, such as BHQ1, BHQ2 or MGB.

[0028] In a specific embodiment, the 3' end of the probe is BHQ1.

[0029] In a specific embodiment, the 3' end of the probe is BHQ2.

[0030] The dosage of a single primer in the composition is 0.05 μM-5 μM; the dosage of a single probe in the composition is 0.05 μM-5 μM.

[0031] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0032] In a particular embodiment, the ingredients of the composition of the invention are present in a package.

[0033] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0034] In a second aspect, the present invention provides the use of the above-mentioned composition of the present invention in the preparation of a kit for simultaneously detecting three common urinary tract infection pathogens and two drug resistance genes, wherein the three common urinary tract infection pathogens are Mycoplasma hominis, Ureaplasma urealyticum and Actinomyces steadiensis; and the two drug resistance genes are the fluoroquinolone resistance gene GyrA and the tetracycline resistance gene tetB.

[0035] Furthermore, the kit is a fluorescent PCR detection kit.

[0036] In a third aspect, the present invention provides a kit for simultaneously detecting three common urinary tract infection pathogens and two drug-resistant genes, wherein the kit comprises the above-mentioned composition of the present invention.

[0037] Furthermore, the kit also includes a fluorescent reaction solution, which includes UNG enzyme, DNA polymerase, dNTPs (U), Mg 2+ At least one of .

[0038] Furthermore, the fluorescent reaction solution also includes Tris-HCl, KCl, and NaCl2.

[0039] In a specific embodiment, the fluorescent reaction solution includes PCR buffer, UNG enzyme, DNA polymerase, dNTPs (U), MgCl2, and DEPC water.

[0040] Common PCR buffers are composed of Tris-HCl, KCl, NaCl2 and other buffer systems. The total volume in a single PCR reaction tube is generally 20-200μl.

[0041] Furthermore, the kit also includes a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0042] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0043] 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 fragment plasmid or fragment DNA or fragment RNA of Mycoplasma hominis, Ureaplasma urealyticum and Actinomycetemcomitans, fluoroquinolone resistance gene GyrA, and tetracycline resistance gene tetB.

[0044] In a fourth aspect, a composition is provided for preparing a detection kit for simultaneously detecting three common urinary tract infection pathogens and two drug resistance genes, comprising the following steps:

[0045] S1. Extract or release nucleic acid from the sample to be tested;

[0046] 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;

[0047] S3. Obtain and analyze the results.

[0048] In a specific embodiment, a composition is provided for preparing a detection kit for simultaneously detecting urinary tract infection pathogens and drug resistance genes, wherein the detection comprises the following steps:

[0049] S1. Extract or release nucleic acid from the sample to be tested;

[0050] 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; further, the reaction conditions of the fluorescent quantitative PCR include: UNG enzyme treatment at 50°C, 2 minutes, 1 cycle; pre-denaturation at 95°C, 5 minutes, 1 cycle; denaturation at 95°C, 15 seconds, extension at 60°C, 35 seconds, 45 cycles.

[0051] S3. Obtain and analyze the results. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 is the test result of the composition of the present invention;

[0053] Figure 2 is the sensitivity test result of the composition of the present invention;

[0054] Figure 3 is the specific detection result of the composition of the present invention;

[0055] Figure 4 The test result of the composition of the present invention on a single test of a Ureaplasma urealyticum positive sample;

[0056] Figure 5 The test results of the composition of comparative example 1 of the present invention on a single test of a Ureaplasma urealyticum positive sample;

[0057] Figure 6 The results are the test results of mixed testing of the composition of comparative example 1 of the present invention on positive samples of Mycoplasma hominis, Ureaplasma urealyticum, Actinobacillus steutzeri, fluoroquinolone resistance gene GyrA, and tetracycline resistance gene tetB of the present application. DETAILED DESCRIPTION

[0058] 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.

[0059] Example 1: Primers and probes used in the present invention

[0060] The primers and probes used in the present invention are shown in Table 1 below:

[0061] Table 1:

[0062]

[0063]

[0064] Among them, the fluorescent group of the probe of Mycoplasma hominis is FAM; the fluorescent group of the probe of Ureaplasma urealyticum is HXE; the fluorescent group of the probe of Actinocytobacterium stuartii is ROX; the fluorescent group of the probe of fluoroquinolone resistance gene Gyr A is CY5; and the fluorescent group of the probe of tetracycline resistance gene tetB is Quasar705.

[0065] Example 2: Method for simultaneously detecting three common urinary tract infection pathogens and two drug resistance genes

[0066] (1) Reagent preparation:

[0067] According to the number of samples to be tested, positive controls, and negative controls, take the corresponding amount of PCR buffer and enzyme mixture according to the ratio (PCR buffer 14.5μL / person + enzyme mixture 1.5μL / person), mix thoroughly to form a PCR mixture, and set aside.

[0068] Sterile saline was used as the negative control matrix, and mixed plasmids containing the corresponding specific gene target sequence were used as the positive control.

[0069] Preparation of enzyme mixture:

[0070] The enzyme mixture consists of UNG enzyme and H-Taq enzyme (DNA polymerase). H-Taq enzyme (5U / μL) and UNG enzyme (2U / μL) are mixed in a certain ratio (1.4μL H-Taq enzyme and 0.1μL UNG enzyme per person). The enzyme mixture is used in an amount of 1.5μL per person.

[0071] (2) Sample processing and sample addition

[0072] Take 300 μL of the sample to be tested, negative control, and positive control into a 1.5 mL centrifuge tube, and use the nucleic acid extraction or purification reagent commercially available from Shengxiang Biotechnology Co., Ltd. (conventional nucleic acid extraction or purification reagents are also acceptable) to extract nucleic acid according to its instructions. Pipette 4 μL of the above-treated sample, negative control, and positive control and add them to the reaction tubes respectively, add 16 μL of PCR mixture to each reaction tube and cover the tube.

[0073] The real-time fluorescence PCR reaction system is configured according to Table 2 below:

[0074] Table 2:

[0075]

[0076]

[0077] (3) The amplification reaction procedure is shown in Table 3:

[0078] Table 3:

[0079]

[0080] (4) Detection

[0081] If the FAM, HEX, CY5, ROX and Quasar705 channels have obvious S-shaped amplification curves and the Ct value is ≤38, it is judged as positive;

[0082] If there is no amplification curve (No Ct) or the Ct value is greater than 38 in the FAM, HEX, ROX, and Quasar705 channels, and CY5 is ≤38, it is considered negative;

[0083] If there is no amplification curve (No Ct) or the Ct value is >38 for the FAM, HEX, ROX and Quasar705 channels, and CY5 is >38, the result is unreliable and needs to be retested;

[0084] The details are shown in Table 4:

[0085] Table 4:

[0086]

[0087] Example 3: Test results of the test samples of the composition of the present invention

[0088] The primers and probes shown in Example 1 were used according to the method of Example 2 to detect and differentiate the mixed samples of Mycoplasma hominis, Ureaplasma urealyticum, Actinobacillus steutzeri, fluoroquinolone resistance gene GyrA, and tetracycline resistance gene tetB. The detection results are as follows: Figure 1 As shown. Figure 1 It can be seen that the composition of the present invention can jointly detect and distinguish Mycoplasma hominis, Ureaplasma urealyticum, Actinobacillus steutzeri, fluoroquinolone resistance gene GyrA, and tetracycline resistance gene tetB, and its detection results are consistent with the Sanger sequencing results of the mixed samples.

[0089] Example 4: Sensitivity of the composition of the present invention

[0090] Each virulence gene was tested for LOD (sensitivity), and the results are shown in Table 5 and Figure 2 As shown in Table 5 and Figure 2It can be seen that the test results show that the composition can still accurately detect samples as low as 1000 copies / mL in each channel, indicating that the sensitivity of the composition of the present invention is 1000 copies / mL.

[0091] Table 5:

[0092]

[0093] Example 5. Specificity of the composition of the present invention

[0094] Cross-reaction: Samples with homology in nucleic acid sequences and other pathogens that may cause the same or similar urinary tract infections (Staphylococcus aureus, Escherichia coli, Proteus, Neisseria gonorrhoeae, Mycoplasma, Chlamydia, Klebsiella, Citrobacter, Enterococcus faecalis and Pseudomonas aeruginosa, etc.) are used as test samples for specific detection. The results are as follows: Figure 3 As shown, from Figure 3 It can be seen that the specificity of the composition of the present invention is good.

[0095] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0096] Due to the principle of complementary base pairing, dimers will form between primers and (or) probes, but this probability is very small and can be ruled out at the beginning of the design. However, when multiple pathogens are detected together, there are many primers and probes, and dimers are likely to occur between primers and primers, probes and probes, or primers and probes. To ensure the conservatism of the design (conservatism is crucial to the accuracy of the test), and to consider the mutual interference between different primers and probes, the primers and probes need to be carefully designed.

[0097] In the present invention, the combination of each primer and probe is irreplaceable and replaceable. Other primers and probes are used to form a different detection system, that is, the Ureaplasma urealyticum primers and probes are replaced (see Table 6, the upstream and downstream primers and probes are shown in SEQ ID NOs: 16 to 18, respectively), and the Ureaplasma urealyticum positive samples are tested separately before and after the replacement of primers and probes. The specific detection results are as follows: Figure 4-5 The detection system after replacing the primers and probes was also used to detect the specific genes of five reproductive pathogens (mixed detection). The specific detection results are shown in Figure 6 As shown, Figure 6 and Figure 1 By comparison, it can be seen that in the amplification curve of the detection system of this comparative example, the performance of Mycoplasma hominis is reduced, the Ct value is delayed, and the fluorescence increment decreases.

[0098] Table 6:

[0099]

[0100] 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 three common urinary tract infection pathogens and two drug resistance genes, characterized in that: The composition comprises at least 4 sets of the following primers and probes: Upstream and downstream primers and probes for detecting Mycoplasma hominis as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Ureaplasma urealyticum as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Actinocoryneformis as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting the fluoroquinolone resistance gene GyrA as shown in SEQ ID NOs: 10 to 12; The upstream and downstream primers and probes for detecting the tetracycline resistance gene tetB are shown in SEQ ID NOs: 13-15.

2. The composition according to claim 1, characterized in that The composition includes 5 sets of primers and probes for detecting: Upstream and downstream primers and probes for detecting Mycoplasma hominis as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Ureaplasma urealyticum as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Actinocoryneformis as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting the fluoroquinolone resistance gene GyrA as shown in SEQ ID NOs: 10 to 12; The upstream and downstream primers and probes for detecting the tetracycline resistance gene tetB are shown in SEQ ID NOs: 13-15.

3. The composition according to claim 1 or 2, 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 group of the probe shown in SEQ ID NO: 3 is FAM; The fluorescent group of the probe shown in SEQ ID NO: 6 is HXE; The fluorescent group of the probe shown in SEQ ID NO: 9 is ROX; The fluorophore of the probe shown in SEQ ID NO: 12 is CY5; The fluorescent group of the probe shown in SEQ ID NO: 15 is Quasar705.

5. The composition according to any one of claims 1 to 4, characterized in that The composition is present in a mixed form.

6. Use of the composition according to claims 1 to 5 in preparing a kit for simultaneously detecting three common urinary tract infection pathogens and two drug resistance genes, characterized in that: The three common urinary tract infection pathogens are Mycoplasma hominis, Ureaplasma urealyticum and Actinomyces steadiensis; the two drug-resistant genes are the fluoroquinolone resistance gene GyrA and the tetracycline resistance gene tetB.

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

8. A kit for simultaneously detecting three common urinary tract infection pathogens and two 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 9, characterized in that The kit also includes a fluorescent reaction solution, which includes Tris-HCl, KCl, NaCl2, UNG enzyme, DNA polymerase, dNTPs (U), Mg 2+ At least one of .

10. Use of a composition for preparing a detection kit for simultaneously detecting three common urinary tract infection pathogens and two drug resistance genes, comprising 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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