Primers and probes, kits and methods of using kits for detection of microorganisms in the urogenital tract

Abstract

The application discloses a primer and probe for urogenital tract microorganism detection, a kit and a use method of the kit. The primer and probe for urogenital tract microorganism detection, the kit and the use method of the kit can simultaneously detect chlamydia trachomatis, ureaplasma urealyticum and gonococcus. By using real-time fluorescent PCR amplification technology, only one nucleic acid extraction and one-step PCR detection reaction are needed. The use method of the kit is rapid, simple, specific, high in sensitivity and wide in detection range. The sensitivity can reach 400 copies / ml, the detection range is 400 copies / ml to 4.00E+09 copies / ml, and the non-chlamydia trachomatis, ureaplasma urealyticum and gonococcus pathogenic microorganisms cannot be detected, so the problem of false positive is not prone to occur.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology detection, in particular to a primer and probe for detecting microorganism in urogenital tract, a kit and a method for using the kit. BACKGROUND

[0002] The research of microorganism in urogenital tract has important clinical and scientific value. There are many kinds of microorganism in urogenital tract, and the common ones are Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU) and Neisseria gonorrhoeae (NG) etc.

[0003] Chlamydia trachomatis is a kind of prokaryotic microorganism with unique development cycle and strict intracellular parasitism, and is a kind of special microorganism between virus, bacteria and rickettsia, belonging to the domain of bacteria, the class of rickettsia, the order of chlamydia and the genus of chlamydia. Chlamydia trachomatis is the most common microorganism causing urogenital infection at home and abroad, and the genital chlamydia infection ranks the third in STD in China, and its incidence rate has a trend of increasing year by year. After the body is infected with Chlamydia trachomatis, specific cellular immunity and humoral immunity can be induced, but the immunity is usually not strong and maintains for a short time, thus causing persistent infection, latent infection and repeated infection.

[0004] Ureaplasma urealyticum, also known as ureaplasma urealyticum, belongs to the genus of ureaplasma in the order of mycoplasma in the class of mollicutes, and is a kind of smallest prokaryotic microorganism between bacteria and virus, mainly distributed in human urogenital tract, and can be transmitted through sexual contact, and can also be vertically transmitted from mother to fetus. The most common parasitic site in male genital tract is urethral orifice and semen, and the most common parasitic site in female genital tract is vagina. Ureaplasma urealyticum is one of the main pathogens of nongonococcal urethritis, and can cause various genital tract inflammations after infection. In men, it can cause nongonococcal urethritis, acute epididymitis, prostatitis, etc.; in women, it can cause endometritis, salpingitis, oophoritis, etc., and intrauterine infection can cause adverse consequences such as miscarriage, stillbirth and premature birth.

[0005] Neisseria gonorrhoeae was discovered by Neisser in 1879, commonly known as gonococcal or gonococcus, is a pathogenic pathogen causing human gonorrhea, belonging to the genus of Neisseria. Neisseria gonorrhoeae has strict parasitism on human body and strong adaptability and invasion ability to human body, and its main pathogenic substances include pilus, outer membrane protein, protease, lipopolysaccharide, etc., and is one of the sexually transmitted diseases with high incidence in developing countries. Neisseria gonorrhoeae mainly infects directly through sexual contact, can also infect indirectly by contacting the clothes or toilet of the patient, and can also infect the fetus during delivery through the birth canal.

[0006] Currently, the laboratory detection methods of Chlamydia trachomatis, Ureaplasma urealyticum and Neisseria gonorrhoeae mainly include isolation and culture method, immunological method, molecular biological method based on nucleic acid detection, etc. The culture method has high specificity and sensitivity, but it has defects such as long clinical detection time (at least 24-48 hours or even longer), complicated process, need of special culture medium and easy influence by miscellaneous bacteria, etc., and is not suitable for large-scale detection. The immunological method is simple and fast, but has poor specificity and low sensitivity. In recent years, the polymerase chain reaction (PCR) method gradually shows its advantages in detection. The fluorescent PCR technology is a more sensitive, specific and accurate nucleic acid detection technology based on traditional PCR technology and combined with spectrum technology, which has accurate detection results, high repeatability, can dynamically respond to pathogen changes and clinical relationship, and avoids the need for post-processing in traditional PCR, reducing pollution.

[0007] Currently, there are some reagent kit products for detecting Chlamydia trachomatis, Ureaplasma urealyticum or Neisseria gonorrhoeae DNA based on real-time fluorescent quantitative PCR technology, but they generally have poor specificity and low sensitivity, and most of them are single detection reagent kits for Chlamydia trachomatis, Ureaplasma urealyticum or Neisseria gonorrhoeae, and the detection effect for mixed detection of multiple pathogenic microorganisms is particularly poor, and the processing process is complicated. SUMMARY

[0008] Therefore, it is necessary to provide a primer and probe, a kit and a use method of the kit for detecting urogenital microorganisms with good specificity and high sensitivity.

[0009] A primer and probe for detecting urogenital microorganisms, comprising a Chlamydia trachomatis amplification primer pair and a corresponding detection probe, a Ureaplasma urealyticum amplification primer pair and a corresponding detection probe, and a Neisseria gonorrhoeae amplification primer pair and a corresponding detection probe; the sequences of the two primers of the Chlamydia trachomatis amplification primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and the sequence of the corresponding detection probe is shown in SEQ ID NO: 3; the sequences of the two primers of the Ureaplasma urealyticum amplification primer pair are shown in SEQ ID NO: 4 and SEQ ID NO: 5 respectively, and the sequence of the corresponding detection probe is shown in SEQ ID NO: 6; the sequences of the two primers of the Neisseria gonorrhoeae amplification primer pair are shown in SEQ ID NO: 7 and SEQ ID NO: 8 respectively, and the sequence of the corresponding detection probe is shown in SEQ ID NO: 9; the two ends of each detection probe are respectively labeled with a fluorescent reporter group and a fluorescent quencher group, and the fluorescent reporter groups of different detection probes are different.

[0010] In one of the embodiments, the fluorescent reporter group labeled on the detection probe of the Chlamydia trachomatis is HEX or VIC, and the fluorescent quencher group is BHQ1; the fluorescent reporter group labeled on the detection probe of the Ureaplasma urealyticum is FAM, and the fluorescent quencher group is BHQ1; the fluorescent reporter group labeled on the detection probe of the Neisseria gonorrhoeae is CY5, and the fluorescent quencher group is BHQ2.

[0011] The kit for detecting the microorganism in the urogenital tract comprises the primer and probe for detecting the microorganism in the urogenital tract.

[0012] In one of the embodiments, the kit for detecting the microorganism in the urogenital tract further comprises an internal standard positive control and its amplification primer pair and detection probe; the internal standard positive control contains a gene fragment of the beta globin family with the sequence shown in SEQ ID NO: 13; the sequences of the two primers of the internal standard positive control amplification primer pair are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the sequence of the corresponding detection probe is shown in SEQ ID NO: 12; the two ends of the detection probe of the internal standard positive control are also labeled with a fluorescent reporter group and a fluorescent quencher group, respectively, and the labeled fluorescent reporter group is different from the fluorescent reporter group labeled on the detection probe of the microorganism in the urogenital tract.

[0013] In one of the embodiments, the fluorescent reporter group labeled on the detection probe of the internal standard positive control is ROX, and the fluorescent quencher group is BHQ2.

[0014] In one of the embodiments, the kit for detecting the microorganism in the urogenital tract comprises inactivated Chlamydia trachomatis, Ureaplasma urealyticum and Neisseria gonorrhoeae positive control.

[0015] In one of the embodiments, the kit for detecting the microorganism in the urogenital tract further comprises at least one of a nucleic acid extraction reagent, a PCR amplification buffer, a DNA polymerase and a dNTPs reagent.

[0016] In one of the embodiments, the dNTPs comprise dATP, dGTP, dTTP, dCTP and dUTP, and the kit further comprises a UNG enzyme.

[0017] A method for using the kit for detecting the microorganism in the urogenital tract, comprising the following steps:

[0018] Extracting the nucleic acid of the sample to be tested to obtain a nucleic acid sample;

[0019] Using the extracted nucleic acid sample as a template, adding the primer and probe for detecting the microorganism in the urogenital tract to perform a real-time fluorescent PCR amplification reaction;

[0020] Detecting the fluorescent signal in the process of real-time fluorescent PCR amplification reaction.

[0021] In one of the embodiments, the method for using the kit for detecting the microorganism in the urogenital tract further comprises the step of using the above-mentioned internal standard positive quality control and / or the above-mentioned positive control to perform the same treatment as the sample to be detected.

[0022] The above-mentioned primer and probe, kit and method for using the kit for detecting the microorganism in the urogenital tract can simultaneously detect chlamydia trachomatis, ureaplasma urealyticum and gonococcus. By using the real-time fluorescent PCR amplification technology, only one nucleic acid extraction and one-step PCR detection reaction are needed. The method for using the kit is fast and simple, and has good specificity. Non-chlamydia trachomatis, ureaplasma urealyticum and gonococcus pathogenic microorganisms cannot be detected, so the problem of false positive is not easy to occur. The sensitivity is high, which can reach 400 copies / ml. The detection range is 400 copies / ml to 4.00E+09 copies / ml, and the detection range is wide.

[0023] Further, the kit and the method for using the kit optimize the combination of the PCR reaction system. By using the characteristics that UNG enzyme can degrade the DNA chain containing dU, UNG enzyme and dUTP are added in the PCR system, which can prevent the pollution of the previous PCR product and prevent the false positive of the sample detection.

[0024] Further, by increasing the internal standard positive control, whether the collected sample is suitable for amplification reaction is evaluated by detecting β-globulin in human epidermal cells, whether the sample to be detected has PCR inhibitors can be monitored, so that the quality of nucleic acid extraction can be evaluated, and the problem of false negative of PCR can be avoided.

[0025] In the process of real-time fluorescent PCR amplification, by real-time fluorescence collection, after the end of PCR amplification, the Ct value and the curve shape can be used to easily judge the positive and negative of chlamydia trachomatis, ureaplasma urealyticum and gonococcus DNA. The detection results can be used for the auxiliary diagnosis of chlamydia trachomatis, ureaplasma urealyticum and gonococcus infection and the observation of drug efficacy, thereby providing reliable experimental basis for the research of chlamydia trachomatis, ureaplasma urealyticum and gonococcus. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figures 1-3 The detection results of the positive control of chlamydia trachomatis, ureaplasma urealyticum and gonococcus are shown in sequence;

[0027] Figure 4 The specific detection results of the negative control;

[0028] Figures 5-7The results of sensitivity detection of positive control samples of different concentrations of Chlamydia trachomatis, different concentrations of Ureaplasma urealyticum and different concentrations of Neisseria gonorrhoeae, in turn, are shown in the following table:

[0029] Figures 8-10 The results of sensitivity detection of positive control samples of Chlamydia trachomatis, Ureaplasma urealyticum and Neisseria gonorrhoeae using different interfering substances, in turn, are shown in the following table:

[0030] Figure 11 and Figure 12 The results of detection of negative control samples without and with dUTP and UNG enzyme, respectively. DETAILED DESCRIPTION

[0031] For the purpose of promoting the understanding of the present application, the present application will be more fully described by reference to the following drawings. The preferred embodiments of the present application are shown in the drawings. However, the present application can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the scope of the application to those skilled in the art.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0033] The primers and probes for detecting microorganisms in the urogenital tract according to an embodiment of the present application include a Chlamydia trachomatis amplification primer pair and a corresponding detection probe, a Ureaplasma urealyticum amplification primer pair and a corresponding detection probe, and a Neisseria gonorrhoeae amplification primer pair and a corresponding detection probe. The sequences of the two primers of the Chlamydia trachomatis amplification primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the sequence of the corresponding detection probe is shown in SEQ ID NO: 3. The sequences of the two primers of the Ureaplasma urealyticum amplification primer pair are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and the sequence of the corresponding detection probe is shown in SEQ ID NO: 6. The sequences of the two primers of the Neisseria gonorrhoeae amplification primer pair are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and the sequence of the corresponding detection probe is shown in SEQ ID NO: 9. Each detection probe is labeled with a fluorescent reporter group and a fluorescent quencher group at both ends, and the fluorescent reporter groups of different detection probes are different.

[0034] In one embodiment, the fluorescent reporter group labeled on the detection probe of each microorganism is located at the 5' end, and the fluorescent quencher group is located at the 3' end. Further, in a specific embodiment, the fluorescent reporter group labeled on the detection probe of Chlamydia trachomatis is HEX or VIC, and the fluorescent quencher group is BHQ1; the fluorescent reporter group labeled on the detection probe of Ureaplasma urealyticum is FAM, and the fluorescent quencher group is BHQ1; and the fluorescent reporter group labeled on the detection probe of Neisseria gonorrhoeae is CY5, and the fluorescent quencher group is BHQ2. It can be understood that, in other embodiments, the fluorescent reporter group labeled on the detection probe of each microorganism is not limited to the above, as long as the fluorescent groups labeled at both ends can undergo fluorescence resonance energy transfer, and the fluorescent reporter group labeled on the detection probe of different microorganisms is different.

[0035] The present embodiment also provides a kit for detecting microorganisms in the urogenital tract, comprising the primers and probes for detecting microorganisms in the urogenital tract described above.

[0036] In one embodiment, the kit for detecting microorganisms in the urogenital tract further comprises an internal standard positive control and its amplification primer pair and detection probe. The internal standard positive control contains a beta globin gene fragment with the sequence shown in SEQ ID NO: 13. In one embodiment, the internal standard positive control is a cloned plasmid containing the above gene fragment. The sequences of the two primers of the internal standard positive control amplification primer pair are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the sequence of the corresponding detection probe is shown in SEQ ID NO: 12. The two ends of the detection probe of the internal standard positive control are also labeled with a fluorescent reporter group and a fluorescent quencher group, and the fluorescent reporter group labeled thereon is different from the fluorescent reporter group labeled on the detection probe of the microorganism in the urogenital tract, such as, in a specific embodiment, the fluorescent reporter group labeled at the 5' end of the detection probe of the internal standard positive control is ROX, and the fluorescent quencher group is BHQ2. Selecting beta globin as the internal standard can monitor sample collection and sample extraction. In one embodiment, the beta globin gene fragment shown in SEQ ID NO: 13 can be amplified by using primers containing restriction endonuclease site sequences and inserted into an empty vector plasmid digested with the same restriction endonuclease, and after transformation of E. coli, positive clones are selected, and the plasmid is extracted to obtain the internal standard positive control.

[0037] Further, in one embodiment, the kit for detecting microorganisms in the urogenital tract comprises inactivated Chlamydia trachomatis, Ureaplasma urealyticum, and Neisseria gonorrhoeae positive control. In a preferred embodiment, the inactivated Chlamydia trachomatis, Ureaplasma urealyticum, and Neisseria gonorrhoeae can be mixed with the cloned plasmid of the internal standard positive control at a predetermined ratio (such as the same concentration) to form a positive reference.

[0038] The positive reference can provide a quantitative control basis for subsequent software analysis. For example, the positive reference can be diluted with TE buffer to four concentrations A, B, C, and D, with concentrations of 1.00-5.00E+07 copies / ml (A), 1.00-5.00E+06 copies / ml (B), 1.00-5.00E+05 copies / ml (C), and 1.00-5.00E+04 copies / ml (D). The subsequent software can automatically obtain the quantitative results of each sample by drawing a standard curve based on the four concentration gradients of the reference.

[0039] Further, the Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, and the internal standard positive control plasmid can be mixed together according to a specific concentration ratio to form a positive control. For example, in a specific embodiment, the Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, and the cloned plasmid of the beta-globulin gene fragment are mixed, and the concentration of each component is 1.00-5.00E+05 copies / ml.

[0040] Further, the kit further includes a negative control. The negative control can be, but is not limited to, sterilized physiological saline and the like.

[0041] In an alternative embodiment, the kit for detecting microorganisms in the urogenital tract further includes at least one of a nucleic acid extraction reagent, a PCR amplification buffer, a DNA polymerase, and dNTPs reagents.

[0042] In a specific embodiment, the 10x PCR amplification buffer includes a 200 mmol / L Tris-HCl solution with a pH of 7.5, a 30 mmol / L magnesium chloride solution, a 500 mmol / L potassium chloride solution, a 0.2% (volume / volume) triton solution, and a 10% (volume / volume) formamide solution.

[0043] The DNA polymerase can be, but is not limited to, a hot-start Taq enzyme, and the use concentration is 1 U / μl-5 U / μl.

[0044] In a specific embodiment, the dNTPs include dATP, dGTP, dTTP, dCTP, and dUTP. Further, the kit further includes UNG enzyme (uracil DNA glycosylase) with a use concentration of 0.05 U / μl-0.2 U / μl. The UNG enzyme has the function of degrading PCR products containing dU, and the use of UNG enzyme and dUTP in the PCR reaction solution can prevent PCR product contamination. Further, in a specific embodiment, the DNA polymerase and the UNG enzyme can be mixed to form an enzyme mixture for use.

[0045] In a preferred embodiment, 5 μl of 10x PCR amplification buffer, dNTPs at a final concentration of 0.2 mmol / L (0.2 mmol / L for each dNTP), 0.2-0.4 μmol / L of each microorganism and the corresponding amplification primers of the positive control clone plasmid, 0.2-0.4 μmol / L of each microorganism corresponding detection probe, and 0.1-0.2 μmol / L of the corresponding detection probe of the clone plasmid are mixed together to form a real-time fluorescent PCR reaction solution, which is then mixed with the nucleic acid extraction at a predetermined ratio for a subsequent co-mixing reaction.

[0046] The present embodiment also provides a method for using a kit for detecting microorganisms in the urogenital tract, which comprises the following steps:

[0047] Extracting nucleic acids from the sample to be tested to obtain a nucleic acid sample;

[0048] Using the extracted nucleic acid sample as a template, adding the primers and probes for detecting microorganisms in the urogenital tract to perform a real-time fluorescent PCR amplification reaction;

[0049] Detecting the fluorescent signal during the real-time fluorescent PCR amplification reaction.

[0050] The conditions of the real-time fluorescent PCR amplification reaction can be determined and adjusted according to the salt ion concentration of the buffer, the length and nucleotide composition of the denatured nucleic acid, the reaction characteristics, and the nucleic acid length, etc. In a preferred embodiment, the following procedure can be used:

[0051]

[0052] In an embodiment, the method for using the kit for detecting microorganisms in the urogenital tract further comprises the step of using the above-mentioned positive control and / or the above-mentioned positive control to perform the same treatment as the sample to be tested.

[0053] Further, in an embodiment, the method for using the kit for detecting microorganisms in the urogenital tract further comprises the step of using a sterile physiological saline solution as a negative control to perform the same treatment as the sample to be tested.

[0054] The primer and probe, the kit and the method for using the kit for detecting the urogenital tract microorganism can simultaneously detect chlamydia trachomatis, ureaplasma urealyticum and gonococcus, and only one nucleic acid extraction and one-step PCR detection reaction are needed by using real-time fluorescent PCR amplification technology. The method for using the kit is fast and simple, has good specificity, cannot detect non-chlamydia trachomatis, ureaplasma urealyticum and gonococcus pathogenic microorganisms, and thus false positive problems are not prone to occur, and the sensitivity is high, can reach 400 copies / ml, the detection range is 400 copies / ml to 4.00E+09 copies / ml, and the detection range is wide.

[0055] Further, the kit and the method for using the kit can prevent sample detection false positive by adding UNG enzyme and dUTP in the PCR system through optimizing the combination of the PCR reaction system and using the characteristics that UNG enzyme can degrade the DNA chain containing dU.

[0056] Further, by adding an internal standard positive control, whether the collected sample is suitable for amplification reaction can be evaluated by detecting β-globulin in human epidermal cells, whether PCR inhibitors exist in the sample to be detected can be monitored, and thus the quality of nucleic acid extraction can be evaluated, and PCR false negative problems can be avoided.

[0057] In the real-time fluorescent PCR amplification process, the real-time fluorescence is collected, and after the PCR amplification is completed, the Ct value and the curve shape can be used to easily judge the positive and negative of chlamydia trachomatis, ureaplasma urealyticum and gonococcus DNA, the detection result can be used for the auxiliary diagnosis of chlamydia trachomatis, ureaplasma urealyticum and gonococcus infection and the observation of drug efficacy, and thus reliable experimental basis is provided for the research of chlamydia trachomatis, ureaplasma urealyticum and gonococcus.

[0058] The following is a specific embodiment part.

[0059] 1. Sample collection

[0060] The sample detected in the embodiment can be urogenital tract secretion.

[0061] Positive control: The cloned plasmids of the gene fragments of chlamydia trachomatis, ureaplasma urealyticum, gonococcus and β-globulin are mixed, and the concentration of each component is 1.00-5.00E+05 copies / ml.

[0062] Negative control: sterilized normal saline.

[0063] Positive reference: inactivated Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, and the above-mentioned internal standard positive control plasmid were mixed at the same concentration to form a positive reference, which was sequentially diluted with TE buffer to A, B, C, D four concentrations, and the concentrations were 1.00-5.00E+07 copies / ml (A), 1.00-5.00E+06 copies / ml (B), 1.00-5.00E+05 copies / ml (C), 1.00-5.00E+04 copies / ml (D).

[0064] 2. Sample nucleic acid extraction

[0065] 2.1 Method one: magnetic bead method for extracting nucleic acid (SuperAll super extraction kit, item number S1006, Hunan Shengxiang Biotechnology Co., Ltd.)

[0066] 1) Virus lysis: add 200 μl-1 ml DNA extraction solution I to each tube, then add 100 μl-1 ml sample to be tested, cover the tube cap, shake and mix for 10 seconds, and centrifuge.

[0067] 2) Magnetic bead adsorption of nucleic acid: add 50 μl-400 μl DNA extraction solution II to each tube, shake and mix for 10 seconds, then stand at room temperature for 5-10 minutes.

[0068] 3) Remove impurities: after centrifugation, place the centrifuge tube on the magnetic bead separator, and slowly suck out the solution after 2-5 minutes.

[0069] 4) Washing: add 400 μl-1 ml DNA extraction solution III and 100 μl-500 μl DNA extraction solution IV to each tube, shake and mix for 3-7 seconds, then centrifuge and place the centrifuge tube on the separator again.

[0070] 5) After 2-5 minutes, the supernatant is divided into two layers, the pipette is inserted into the bottom of the centrifuge tube, and the liquid is slowly sucked out from the bottom and discarded, and after standing for 1-3 minutes, the residual liquid at the bottom of the tube is completely sucked out and discarded.

[0071] 6) Add 50-100 μl DNA elution solution to each tube, use the pipette to elute the brown residue adsorbed on the wall of the centrifuge tube with the DNA elution solution, and repeat several times to completely elute it.

[0072] 7) After standing for 5-10 minutes, place the centrifuge tube on the separator again, and after 2-5 minutes, the eluate is sucked out and placed in a new centrifuge tube for standby.

[0073] 8) Add 5-10 μl of the above pretreated sample to be tested, negative control, and positive control to each reaction tube.

[0074] 9) Add PCR-mix 40-45 μl to each tube, cover the tube cap (after removing the bubble), centrifuge at 2000 rpm for 30 seconds.

[0075] 2.2 Method two: after concentrating the sample by high-speed centrifugation, release the nucleic acid (the sample release agent includes 0.01-0.5 mM / L savan ting, 50-200 mM / L potassium chloride, 0.01%-2% (w / v) sodium dodecyl sulfate, and 0.05%-1% (v / v) ethanol)

[0076] 1) Take 200-500 μl of the sample to be tested, centrifuge at 13,000 rpm for 5 minutes, discard the supernatant, add 20-50 μl of the release agent, stand for 10 minutes, and then prepare for use;

[0077] 2) Add 5-10 μl of the above-mentioned pretreated sample to be tested, negative control, and positive control to each reaction tube;

[0078] 3) Add PCR-mix 40-45 μl to each tube, cover the tube cap (after removing the bubble), centrifuge at 2000 rpm for 30 seconds.

[0079] Note: PCR-mix: take the corresponding amount of real-time fluorescent PCR reaction solution and enzyme mixture according to the proportion (real-time fluorescent PCR reaction solution 38-44 μl / person, enzyme mixture 1-2 μl / person, total volume 40-45 μl / person), mix thoroughly to form PCR-mix, and centrifuge immediately for standby.

[0080] The traditional method mainly uses the boiling method to extract the nucleic acid of chlamydia trachomatis, ureaplasma urealyticum, and gonococcus (i.e., first concentrate and wash the secretion sample, then add a lysis solution, boil, high-speed centrifuge, and take the supernatant as the template). For the concentration step, different institutions often have different concentration effects, some can see the precipitate, and some cannot. Seeing the precipitate is because the virus and protein are concentrated, which will make it difficult to mix thoroughly when adding the lysis solution later, and not being able to see the precipitate will make the operator unable to determine whether the virus nucleic acid will be blown off when discarding the supernatant. The present embodiment compares and optimizes the extraction method of chlamydia trachomatis, ureaplasma urealyticum, and gonococcus DNA, which can complete the release and extraction of DNA without heating, and the extraction process is simple and easy to operate, and does not have the many drawbacks of the traditional boiling method.

[0081] 3. Fluorescent PCR reaction (on a fluorescent quantitative PCR amplifier)

[0082] 1) Place the PCR reaction tube into the sample slot of the amplifier, and set the sample name and positive reference concentration in the corresponding order.

[0083] 2) Fluorescence detection channel selection: 1) Select FAM channel (Reporter: FAM, Quencher: none) to detect Ureaplasma urealyticum DNA; 2) Select HEX or VIC channel (Reporter: HEX / VIC, Quencher: none) to detect Chlamydia trachomatis DNA; 3) Select ROX channel (Reporter: ROX, Quencher: none) to detect internal standard; 4) Select CY5 channel (Reporter: CY5, Quencher: none) to detect Gonococcus DNA; 5) Passive Reference is set as none.

[0084] 3) Fluorescence quantitative PCR reaction conditions are as follows:

[0085]

[0086] 4) Result analysis

[0087] After the reaction is completed, the instrument automatically saves the results, which can be automatically analyzed by using the software of the instrument (manual adjustment of the start value, end value and threshold value of the baseline can also be used for analysis), and then the Ct value and the fixed value of the sample are recorded. The intersection of the amplification curve and the threshold line is called Ct (i.e. cycle threshold, which refers to the cycle number of the fluorescence signal in the PCR reaction tube reaching the set threshold value). The instrument software can automatically obtain the fixed value of each sample according to the Ct value of each sample, through the standard curve drawn by the four concentration gradient quantitative reference products. If the sample amplification curve is S-shaped, there is a Ct value and Ct≤38, it can be judged as Chlamydia trachomatis / Ureaplasma urealyticum / Gonococcus positive; if the sample amplification curve is flat, there is no Ct value (Undet) display, it can be judged as Chlamydia trachomatis / Ureaplasma urealyticum / Gonococcus negative.

[0088] 1) Accuracy: positive control is detected, please refer to Figure 1 , Figure 2 and Figure 3 , the results are all positive and accurate, and there is no cross reaction.

[0089] 2) Specificity: negative control is detected, please refer to Figure 4 , the results are all negative, and the detection results of other pathogens (Herpes simplex virus 2, human papilloma virus 6, human papilloma virus 16, Mycoplasma genitalium, Mycoplasma hominis) with the same infection site are all negative.

[0090] 3) Precision: the coefficient of variation (CV, %) of intra-batch precision Ct value is ≤5%.

[0091] 4) Minimum detection limit: please refer to Figure 5 ,Figure 6 and Figure 7 Positive control samples of Chlamydia trachomatis, Ureaplasma urealyticum, and Neisseria gonorrhoeae at different concentrations were tested. The lowest concentration was 400 copies / ml, and all test results were positive, indicating that the lowest detection limit for Chlamydia trachomatis, Ureaplasma urealyticum, and Neisseria gonorrhoeae can reach 400 copies / ml, which is very sensitive.

[0092] 5) Interfering substances: Please refer to Figure 8 , Figure 9 and Figure 10 Adding 2 g / L hemoglobin or 10% mucin to the sample to be tested has no significant effect on the detection of this primer and probe.

[0093] 6) Comparison Figure 11 and Figure 12 ( Figure 11 The dNTPs used do not contain dUTPs, and UNG enzyme is not used in the detection process. Figure 12 The negative control sample, which used dNTPs containing dUTPs and employed UNG enzyme during the detection process, showed a non-negative result, indicating contamination. Prevention experiments of PCR product contamination demonstrated that adding an appropriate amount of UNG enzyme to the PCR reaction system can prevent PCR product contamination.

[0094] Clinical sample testing shows that the rapid test kit of this invention has a detection range of 400 copies / ml to 4.00E+09 copies / ml, with a wide detection limit and a lower detection limit, i.e., sensitivity, of 400 copies / ml, indicating high sensitivity.

[0095] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0096] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims. sequence list <110> Hunan Shengxiang Biotechnology Co., Ltd. <120> Primers and probes, kits, and instructions for use for urogenital tract microbial testing <130> 111 <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 atgttgcatg atgctttatc 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <400> 2 gaaacggatc taagcttgtc a 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <400> 3 tgacaagctt agatccgttt c 21 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <400> 4 tctgctcgtg aagtattac 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 gaaactagtt tagtaccatc 20 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <400> 6 gttgatcaag ttatggaagg tg 22 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <400> 7 caagtgcgtt aaggctttca t 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 cgcagcaatc gagcagcgaa 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 ttcgctgctc gattgctgcg 20 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <400> 10 gactctctct gcctattggt ctatt 25 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <400> 11 cccataacag catcaggagt g 21 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <400> 12 cagatcccca aagactcaa agaacc 26

Claims

1. Primers and probes for detecting microorganisms in the urogenital tract, characterized in that, The Chlamydia trachomatis amplification primer pair and the corresponding detection probe, the Ureaplasma urealyticum amplification primer pair and the corresponding detection probe, and the Neisseria gonorrhoeae amplification primer pair and the corresponding detection probe; the sequences of the two primers of the Chlamydia trachomatis amplification primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and the sequence of the corresponding detection probe is shown in SEQ ID NO: 3; the sequences of the two primers of the Ureaplasma urealyticum amplification primer pair are shown in SEQ ID NO: 4 and SEQ ID NO: 5 respectively, and the sequence of the corresponding detection probe is shown in SEQ ID NO: 6; the sequences of the two primers of the Neisseria gonorrhoeae amplification primer pair are shown in SEQ ID NO: 7 and SEQ ID NO: 8 respectively, and the sequence of the corresponding detection probe is shown in SEQ ID NO: 9; the two ends of each detection probe are labeled with a fluorescent reporter group and a fluorescent quencher group respectively, and the fluorescent reporter groups of different detection probes are different.

2. The primer and probe for detecting urogenital microorganism according to claim 1, wherein The fluorescent reporter group labeled on the detection probe of the Chlamydia trachomatis is HEX or VIC, and the fluorescent quencher group is BHQ1; the fluorescent reporter group labeled on the detection probe of the Ureaplasma urealyticum is FAM, and the fluorescent quencher group is BHQ1; the fluorescent reporter group labeled on the detection probe of the Neisseria gonorrhoeae is CY5, and the fluorescent quencher group is BHQ2.

3. A kit for detecting a microorganism in a urogenital tract, characterized by comprising: The kit comprises the primer and probe for detecting the microorganism in the urogenital tract as claimed in claim 1 or 2.

4. The kit for detecting urogenital microorganisms as described in claim 3, characterized in that, The kit further comprises an internal standard positive control and its amplification primer pair and detection probe; the internal standard positive control contains a beta globin gene fragment with the sequence shown in SEQ ID NO: 13; the sequences of the two primers of the internal standard positive control amplification primer pair are shown in SEQ ID NO: 10 and SEQ ID NO: 11 respectively, and the sequence of the corresponding detection probe is shown in SEQ ID NO: 12; the two ends of the detection probe of the internal standard positive control are also labeled with a fluorescent reporter group and a fluorescent quencher group respectively, and the fluorescent reporter group labeled thereon is different from the fluorescent reporter group labeled on the detection probe of the microorganism in the urogenital tract.

5. The kit for detecting urogenital microorganisms as described in claim 4, characterized in that, The fluorescent reporter group labeled on the detection probe of the internal standard positive control is ROX, and the fluorescent quencher group is BHQ 2.

6. The kit for detecting a urogenital microorganism according to claim 4 or 5, wherein the sample is a sample of a body fluid obtained from a subject. The kit further comprises inactivated Chlamydia trachomatis, Ureaplasma urealyticum and Neisseria gonorrhoeae positive control; the positive control comprises 1.00-5.00E+05 copies / ml Chlamydia trachomatis, 1.00-5.00E+05 copies / ml Ureaplasma urealyticum, 1.00-5.00E+05 copies / ml Neisseria gonorrhoeae and 1.00-5.00E+05 copies / ml beta globin gene fragment cloned plasmid.

7. The kit for detecting a urogenital microorganism according to claim 4 or 5, wherein the sample is a sample of a body fluid obtained from a urogenital organ. The kit further comprises at least one of nucleic acid extraction reagent, PCR amplification buffer, DNA polymerase and dNTPs reagent.

8. The kit for the detection of urogenital microorganisms according to claim 7, wherein The dNTPs comprise dATP, dGTP, dTTP, dCTP and dUTP, and the kit further comprises UNG enzyme.

9. A method for using a kit for the diagnosis and treatment of non-disease of the urogenital tract microorganism detection, characterized by, The kit comprises the following steps: extracting nucleic acid of the sample to be tested to obtain a nucleic acid sample; The extracted nucleic acid sample is used as a template, and the primer and probe for detecting the microorganism in the urogenital tract as claimed in claim 3 are added to perform real-time fluorescent PCR amplification reaction; The fluorescent signal is detected during the real-time fluorescent PCR amplification reaction.

10. The method for using the kit for detecting urogenital microorganisms according to claim 9, wherein, The steps of treating the sample to be detected in the same way as the positive control sample are also included, using the internal standard positive quality control as claimed in claim 4 or 5 and / or the positive control sample as claimed in claim 6.

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