Primer, probe, kit and detection method for quantitatively detecting nucleic acid of chlamydia trachomatis

Abstract

The invention belongs to the field of molecular biology, and relates to a primer, a probe, a kit and a detection method for quantitatively detecting nucleic acid of chlamydia trachomatis, and the primer comprises an upstream primer for detecting the chlamydia trachomatis, a downstream primer for detecting the chlamydia trachomatis, an upstream primer for detecting an internal control gene and a downstream primer for detecting the internal control gene; the probe comprises a chlamydia trachomatis detection probe and an internal control probe; nucleotide sequences of upstream and downstream primers for detecting chlamydia trachomatis are shown as SEQ ID NO: 1 and SEQ ID NO: 2; nucleotide sequences of upstream and downstream primers for detecting the internal control gene are as shown in SEQ ID NO: 3 and SEQ ID NO: 4; the nucleotide sequence of the probe for detecting the chlamydia trachomatis is as shown in SEQ ID NO: 5, and the nucleotide sequence of the internal control probe is as shown in SEQ ID NO: 6. The method is easy to operate, operation steps are greatly simplified, the detection efficiency is high, and the chlamydia trachomatis can be qualitative and quantitative at the same time; and secondly, the combination characteristic of the primer and the probe is high, and the detection sensitivity and the detection accuracy of the chlamydia trachomatis are high.

Description

Technical Field

[0001] The present application relates to the technical field of molecular biology, and in particular, to a primer and a probe for quantitatively detecting Chlamydia trachomatis nucleic acid, a kit and a detection method thereof. Background Art

[0002] Chlamydia trachomatis (CT) is a type of prokaryotic cell microorganism that strictly parasitizes within eukaryotic cells and has a unique developmental cycle. According to the estimation of the World Health Organization (WHO), in 2008, the number of globally recognized cases of urogenital Chlamydia trachomatis infection reached 105.7 million person-times, and 2 / 3 of these cases occurred in developing countries. Its clinical course is often concealed, protracted, and with mild symptoms. However, Chlamydia trachomatis can cause various diseases, such as trachoma; it is also related to human urogenital diseases, such as non-gonococcal urethritis, vaginitis, cervicitis, endometritis, etc.; Chlamydia trachomatis can also be transmitted from mother to child to cause neonatal conjunctivitis and pneumonia, and can lead to premature rupture of membranes, premature birth, neonatal death, etc. In the initial stage of Chlamydia trachomatis infection, patients usually do not show obvious clinical manifestations, resulting in their failure to seek medical treatment in a timely manner after infection and may be in a state of persistent infection for a long time, seriously endangering their physical health. Therefore, early, rapid, and accurate diagnosis of the infection has positive significance for its treatment and improvement of the prognosis.

[0003] Currently, for the detection of Chlamydia trachomatis, the detection methods in clinical applications mainly include cell culture method, specimen smear staining microscopy, fluorescent antibody and enzyme-linked immunosorbent assay, colloidal gold immunochromatography, serological detection method, fluorescent PCR method, etc. These methods are widely used in clinical practice and play an important role in assisting clinicians to make appropriate treatment plans. However, these methods are all qualitative detections, that is, they can only report the presence or absence of the target pathogen, and cannot meet the needs of quantitative detection. Summary of the Invention

[0004] The purpose of the present application is to provide a primer and a probe for quantitatively detecting Chlamydia trachomatis nucleic acid, a kit and a detection method thereof, so as to solve the technical problem in the prior art that the Chlamydia trachomatis nucleic acid in a sample cannot be quantitatively detected quickly and accurately.

[0005] To solve the above technical problem, the embodiment of the present application provides a primer and a probe for quantitatively detecting Chlamydia trachomatis nucleic acid, and adopts the following technical solutions:

[0006] The primer includes an upstream primer for detecting Chlamydia trachomatis, a downstream primer for detecting Chlamydia trachomatis, an upstream primer for detecting an internal control gene, and a downstream primer for detecting an internal control gene; the probe includes a probe for detecting Chlamydia trachomatis and an internal control probe;

[0007] Among them, the nucleotide sequence of the upstream primer for detecting Chlamydia trachomatis is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer for detecting Chlamydia trachomatis is shown in SEQ ID NO: 2; the nucleotide sequence of the upstream primer for detecting the internal control gene is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer for detecting the internal control gene is shown in SEQ ID NO: 4;

[0008] The nucleotide sequence of the probe for detecting Chlamydia trachomatis is shown in SEQ ID NO: 5, and the nucleotide sequence of the internal control probe is shown in SEQ ID NO: 6.

[0009] Furthermore, the 5' end of the nucleotide sequences corresponding to the probe for detecting Chlamydia trachomatis and the internal control probe is labeled with a fluorescent group; the 3' end of the nucleotide sequences corresponding to the probe for detecting Chlamydia trachomatis and the internal control probe is labeled with a quenching group;

[0010] Among them, the fluorescent group is selected from FAM, HEX, VIC; the quenching group is selected from MGB, BHQ, TAMRA or Eclipse.

[0011] To solve the above technical problems, the embodiments of the present application also provide a kit for quantitatively detecting Chlamydia trachomatis nucleic acid, adopting the following technical solutions:

[0012] It includes a primer-probe mixture, a premix, a CT positive control, and a CT negative control; among them, the primer-probe mixture includes the primers and probes for quantitatively detecting Chlamydia trachomatis nucleic acid as described above.

[0013] Furthermore, the final concentrations of the upstream primer for detecting Chlamydia trachomatis and the downstream primer for detecting Chlamydia trachomatis in the PCR reaction system are 0.50 - 0.70 μmol / L respectively; the final concentration of the probe for detecting Chlamydia trachomatis in the PCR reaction system is 0.20 - 0.30 μmol / L;

[0014] The final concentrations of the upstream primer for detecting the internal control gene and the downstream primer for detecting the internal control gene in the PCR reaction system are 0.40 - 0.60 μmol / L respectively; the final concentration of the internal control probe in the PCR reaction system is 0.20 - 0.30 μmol / L.

[0015] Furthermore, the components of the premix include Tris-HCl buffer, dNTP, and PCR reaction enzyme.

[0016] Furthermore, the components of the CT positive control include inactivated Chlamydia trachomatis culture and pseudovirus containing an internal standard fragment, and the components of the CT negative control include pseudovirus containing an internal standard fragment and TE buffer.

[0017] To solve the above technical problems, an embodiment of the present application further provides a detection method for quantitatively detecting Chlamydia trachomatis nucleic acid. The kit described above is used to detect Chlamydia trachomatis nucleic acid for non-diagnostic purposes, and the following technical solutions are adopted:

[0018] Collect a sample to be tested for Chlamydia trachomatis, extract nucleic acid from the sample to be tested to obtain the nucleic acid of the sample to be tested;

[0019] Take out the primer-probe mixture and the premix from the kit, melt and mix them at room temperature to prepare a PCR reaction solution system, and dispense the PCR reaction solution system into a preset number of PCR reaction tubes;

[0020] Correspondingly add the nucleic acid of the sample to be tested, the CT positive control product, and the CT negative control product to the PCR reaction tubes, transfer them to the droplet preparation area after instantaneous centrifugation;

[0021] Use a droplet preparation chip to prepare droplets of the liquid in the PCR reaction tubes, and perform ddPCR amplification reaction on the prepared droplets according to the PCR amplification conditions to obtain amplification products;

[0022] Perform quantitative analysis on the amplification products to obtain the copy number of Chlamydia trachomatis nucleic acid in the sample to be tested.

[0023] Further, the sample to be tested for Chlamydia trachomatis uses a human urogenital system sample.

[0024] Further, the detection method further includes:

[0025] Take the sample to be tested for Chlamydia trachomatis with a concentration of (1.15 - 1.20)×10 7 copies / mL, and use a negative sample to dilute the sample to be tested at 1:10, 1:10 2 、1:10 3 、1:10 4 and 1:10 5 to obtain samples to be tested with different dilution concentrations, and the sample numbers are S1 - S5;

[0026] Extract nucleic acid from the samples to be tested with different dilution concentrations S1 - S5 to obtain the nucleic acids of the samples to be tested corresponding to different dilution concentrations.

[0027] Further, the PCR amplification conditions are as follows:

[0028] Pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing and extension at 58°C for 1 min, for a total of 45 cycles; then, enzyme inactivation at 98°C for 10 min; finally, hold at 4°C.

[0029] Compared with the prior art, the present application mainly has the following beneficial effects:

[0030] The present application provides a primer, a probe, a kit and a detection method for quantitatively detecting Chlamydia trachomatis nucleic acid. Based on digital PCR technology, the Chlamydia trachomatis nucleic acid in a sample can be quantitatively detected, and samples with a concentration as low as 1000 copies / mL can be stably detected. The operation is simple, greatly simplifying the operation steps, and the detection efficiency is high. It can simultaneously perform qualitative and quantitative detection of Chlamydia trachomatis. Secondly, the combination characteristics of the primer and probe of the present application are high, and the detection sensitivity and detection accuracy for Chlamydia trachomatis are high. In addition, the present application does not require setting a standard curve. According to the results of the fluorescence type and the number of fluorescent droplets, the positive and negative of Chlamydia trachomatis and the copy number can be directly judged, and an accurate quantitative detection result of Chlamydia trachomatis can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the solutions in the present application, the following will briefly introduce the drawings required for the description of the embodiments of the present application. Obviously, the following drawings are some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0032] Figure 1 It is a schematic diagram of the PCR reaction result of the CT negative control product detected by the kit of the present application;

[0033] Figure 2 It is a schematic diagram of the PCR reaction result of the CT positive control product detected by the kit of the present application;

[0034] Figures 3 to 7 They are schematic diagrams of the detection results of the samples numbered S1 to S5 detected by the kit of the present application in sequence;

[0035] Figure 8 It is a schematic diagram of the detection results of the positive reference products PC01 to PC10 detected by the kit of the present application;

[0036] Figure 9 It is a schematic diagram of the detection results of the negative reference products NC01 to NC9 detected by the kit of the present application;

[0037] Figure 10 It is the detection situation of the kit of the present application for the positive samples numbered 1 - 10;

[0038] Figure 11 It is the detection situation of the kit of the present application for the positive samples numbered 11 - 20;

[0039] Figure 12 It is the detection situation of the kit of the present application for the negative samples numbered 21 - 30;

[0040] Figure 13 This is the detection result of the kit of the present application for negative samples numbered 31 - 40. Detailed implementation manners

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs; the terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "comprising" and "having" and any variations thereof in the specification and claims of this application and the above drawings are intended to cover non - exclusive inclusion. The terms "first", "second", etc. in the specification and claims of this application or the above drawings are used to distinguish different objects and not to describe a specific order.

[0042] Reference to "embodiment" herein means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of this application. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0043] To enable those skilled in the art to better understand the solution of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the drawings.

[0044] The primers, probes, kit and detection method for quantitatively detecting Chlamydia trachomatis nucleic acid provided by this application adopt the droplet digital PCR (hereinafter referred to as "ddPCR") technology to quantitatively detect the Chlamydia trachomatis nucleic acid in the extracted urogenital system samples, and have the advantages of simple operation, being able to perform qualitative and quantitative detection simultaneously, and high detection efficiency.

[0045] Among them, digital PCR (dPCR) is a test method that can perform absolute quantification on target fragments in a sample to be tested. Digital PCR will disperse a fluorescence PCR reaction system into tens of thousands of micro-reaction systems and then perform PCR. Each micro-reaction is parallel to each other. The target fragment templates of the sample to be tested will also be dispersed into each micro-reaction system accordingly. If the number of dispersed micro-reaction systems is large enough, then finally there will be only 1 or 0 fragment templates in each micro-reaction system, thus realizing "single-molecule template PCR amplification". Micro-reactions with 1 or more target fragments will generate fluorescence after PCR and are positive units, while micro-reactions without target fragments will not generate fluorescence after PCR and are negative units. By calculating the number of positive and negative units and combining statistical formulas, the copy number of the target fragment in the original sample can be calculated. In this application, the target fragment is the DNA of Chlamydia trachomatis pathogen or a DNA fragment of Chlamydia trachomatis pathogen.

[0046] Based on the digital PCR technology, this application proposes a primer and a probe for quantitatively detecting Chlamydia trachomatis nucleic acid. The primer includes an upstream primer for detecting Chlamydia trachomatis (i.e., CT upstream primer), a downstream primer for detecting Chlamydia trachomatis (i.e., CT downstream primer), an upstream primer for detecting internal control gene (i.e., internal control upstream primer), and a downstream primer for detecting internal control gene (i.e., internal control upstream primer); the probe includes a probe for detecting Chlamydia trachomatis (CT detection probe) and an internal control probe.

[0047] The length of the primer is generally between 15 and 30 bases. The commonly used primer length is 18 - 27bp, but it should not be greater than 38, because too long will cause its extension temperature to be greater than 74°C, which is not suitable for Taq DNA polymerase to react. The length of the PCR amplification product should not be too large, generally between 80 - 300bp. The 4 bases between primers should be randomly distributed to avoid the continuous arrangement of more than 4 identical bases. The GC content is preferably between 40% and 60%. Avoid palindromic or complementary sequences of more than 4bp between the primer itself and primers, and there should be no complementary bases at the 3' end. The annealing temperature is generally selected between 55°C and 60°C, because this temperature can affect the specificity of PCR. For most primers, the difference between the annealing temperature and the Tm value does not exceed 2 - 3°C. The length of the probe should be between 15 - 45bp (preferably 20 - 30bp) to ensure binding specificity. The Tm value is generally between 65 - 70°C, usually 5 - 10°C higher than the primer Tm value, and the GC content is between 40% - 70%. After the primer and probe design is completed, its specificity should be detected by BLAST.

[0048] Primers were designed for the detection site of the human OmpA gene. A pair of specific PCR primers (i.e., the upstream and downstream primers for detecting Chlamydia trachomatis) and a hybridization probe (i.e., the probe for detecting Chlamydia trachomatis) were designed for this detection site respectively, which were used to hybridize the amplified products of the OmpA gene detection site. At the same time, a pair of amplification primers (i.e., the upstream and downstream primers for detecting the internal control gene) and a hybridization probe (i.e., the internal control probe) were designed according to the sequence of the internal control gene.

[0049] Primer design was carried out first, and a large number of experiments were conducted to screen the designed specific primers and probes, and they were combined, optimized and verified. By exploring reaction conditions and optimizing methods, the advantages and disadvantages of linearity, specificity, sensitivity and repeatability among different primers and probes were compared. Finally, the optimal primer-probe combination with no mutual interference, high amplification efficiency and good specificity after combination was selected. Among them, the sequences of the designed primers and probes are shown in Table 1.

[0050] Table 1 Nucleotide sequence information of primers and probes for quantitative detection of Chlamydia trachomatis nucleic acid

[0051]

[0052]

[0053] According to the above table, the nucleotide sequence of the upstream primer for detecting Chlamydia trachomatis is shown as SEQ ID NO: 1, and the nucleotide sequence of the downstream primer for detecting Chlamydia trachomatis is shown as SEQ ID NO: 2; the nucleotide sequence of the upstream primer for detecting the internal control gene is shown as SEQ ID NO: 3, and the nucleotide sequence of the downstream primer for detecting the internal control gene is shown as SEQ ID NO: 4; the nucleotide sequence of the probe for detecting Chlamydia trachomatis is shown as SEQ ID NO: 5, and the nucleotide sequence of the internal control probe is shown as SEQ ID NO: 6.

[0054] Both ends of the above probes are labeled with a fluorescent group and a quenching group. Specifically, the 5' end of the nucleotide sequences corresponding to the probe for detecting Chlamydia trachomatis and the internal control probe is labeled with a fluorescent group; the 3' end of the nucleotide sequences corresponding to the probe for detecting Chlamydia trachomatis and the internal control probe is labeled with a quenching group.

[0055] Among them, the fluorescent group is selected from FAM, HEX, VIC; the quenching group is selected from MGB, BHQ, TAMRA or Eclipse.

[0056] The fluorescent groups labeled at the 5'-ends of the probe for detecting Chlamydia trachomatis and the internal control probe are different, so as to obtain the detection results according to different fluorescence channels. Among them, the 5'-end of the probe for detecting Chlamydia trachomatis is labeled with the FAM fluorescent group, and the 3'-end of the probe for detecting Chlamydia trachomatis is labeled with the MGB quenching group; the 5'-end of the internal control probe is labeled with the VIC fluorescent group, and the 3'-end of the internal control probe is labeled with the MGB quenching group.

[0057] The primer for detecting Chlamydia trachomatis and the probe for detecting Chlamydia trachomatis labeled with the FAM fluorescent group can be used for the quantitative detection of Chlamydia trachomatis. According to the presence or absence of positive droplets in the FAM channel and the number of positive and negative droplets, it can be judged whether Chlamydia trachomatis is contained in the sample and the concentration of the target fragment of Chlamydia trachomatis in the sample can be calculated.

[0058] This application has specifically optimized the primer and probe combination for detecting Chlamydia trachomatis, reduced the misdiagnosis probability caused by non-specific binding, and designed an internal control gene detection system for monitoring and correction.

[0059] This application also provides a kit for quantitatively detecting Chlamydia trachomatis nucleic acid, which includes a primer-probe mixture, a premix, a CT positive control product and a CT negative control product; wherein, the primer-probe mixture includes the primer and probe for quantitatively detecting Chlamydia trachomatis nucleic acid as described above.

[0060] Among them, the final concentrations of the upstream primer for detecting Chlamydia trachomatis and the downstream primer for detecting Chlamydia trachomatis in the PCR reaction system are 0.50 - 0.70 μmol / L respectively; the final concentration of the probe for detecting Chlamydia trachomatis in the PCR reaction system is 0.20 - 0.30 μmol / L; the final concentrations of the upstream primer for detecting the internal control gene and the downstream primer for detecting the internal control gene in the PCR reaction system are 0.40 - 0.60 μmol / L respectively; the final concentration of the internal control probe in the PCR reaction system is 0.20 - 0.30 μmol / L.

[0061] Preferably, the final concentration of the upstream primer for detecting Chlamydia trachomatis in the PCR reaction system is 0.60 μmol / L, the final concentration of the downstream primer for detecting Chlamydia trachomatis in the PCR reaction system is 0.60 μmol / L; the final concentration of the probe for detecting Chlamydia trachomatis in the PCR reaction system is 0.25 μmol / L; the final concentration of the upstream primer for detecting the internal control gene in the PCR reaction system is 0.50 μmol / L, the final concentration of the downstream primer for detecting the internal control gene in the PCR reaction system is 0.50 μmol / L; the final concentration of the internal control probe in the PCR reaction system is 0.25 μmol / L.

[0062] The components of the premixed solution include Tris-HCl buffer, dNTP, and PCR reaction enzymes. The PCR reaction enzymes include Taq DNA polymerase, etc.

[0063] The premixed solution can be the ddPCR Supermix for Probes (No dUTP) provided by Bio-rad for DNA sample droplet digital PCR, with the product numbers: 1864023 or 1864024.

[0064] The kit of this application also includes control products, which include CT positive control products and CT negative control products. Among them, the components of the CT positive control product include inactivated Chlamydia trachomatis culture and pseudovirus containing internal standard fragments, and the components of the CT negative control product include pseudovirus containing internal standard fragments and TE buffer.

[0065] Among them, in the CT positive control product, the concentration of inactivated Chlamydia trachomatis culture is 3×10^5 copies / mL, and the concentration of pseudovirus containing internal standard fragments is 7×10 4 copies / mL; in the CT negative control product, the concentration of pseudovirus containing internal standard fragments is 1×10 5 copies / mL, and the TE buffer is 1×TE.

[0066] The detection samples applicable to the kit of this application are human urogenital system samples.

[0067] The standard for the kit of this application to determine the detection effectiveness is:.

[0068] In each detection, only the reaction wells with the number of droplets ≥ 10000 in all reaction wells are valid reaction wells;

[0069] Among the valid reaction wells, the negative control group and the positive control group should be included. When the detection result of the positive control group is positive, the detection result of the negative control group is negative, and the concentration detection result of the positive control group is 3×10 5 ±10% copies / mL, the detection result of this time is valid.

[0070] Based on the above-mentioned kit for quantitatively detecting Chlamydia trachomatis nucleic acid, this application also provides a method for quantitatively detecting Chlamydia trachomatis nucleic acid for non-diagnostic purposes by using the kit as described above, including the following steps:

[0071] Step S10, collect the Chlamydia trachomatis sample to be tested, extract the nucleic acid from the sample to be tested to obtain the nucleic acid of the sample to be tested.

[0072] The Chlamydia trachomatis sample to be tested is a human urogenital system sample, including male urinary tract samples and female genital tract samples.

[0073] Male urinary tract sample: Insert a cotton swab 2 - 3 cm deep into the urethra, rotate for 3 - 5 seconds and then take it out. Place the cotton swab in a sample tube containing cell preservation solution. Break off the excess brush handle at the tube opening, keep the brush head in the sample tube, tighten the tube cap, and label the sample properly.

[0074] Female genital tract sample: Take samples from the cervix. First, use a cotton swab to remove the mucus at the cervical orifice, then insert another cotton swab 1.0 cm - 1.5 cm into the cervical orifice, rotate and stay for 10 - 30 seconds to allow the cotton swab to fully absorb the secretions and exfoliated cells. Place the two cotton swabs in a sample tube containing cell preservation solution. Break off the excess brush handle at the tube opening, keep the brush head in the sample tube, tighten the tube cap, and label the sample properly.

[0075] Use a qualified human urogenital system extraction kit to extract the sampling solution. For example, use the nucleic acid extraction or purification reagent (registration number: Yue Sui Xie Bei 20170583) produced by Daan Gene Co., Ltd. to extract and purify the nucleic acid from the collected human urogenital system samples.

[0076] Step S20: Take out the primer - probe mixture and the premix from the kit, melt them at room temperature and mix well to prepare the PCR reaction solution system, and aliquot the PCR reaction solution system into a preset number of PCR reaction tubes.

[0077] Take out the primer - probe mixture and the premix from the kit, melt them at room temperature and mix well with a vortex oscillator. After centrifuging at 8000 rpm - 10000 rpm for 10 - 20 s, prepare the PCR reaction system, and aliquot the PCR reaction system into a preset number of PCR reaction tubes.

[0078] Among them, the preset number N = the number of test samples + CT negative control + CT positive control

[0079] The single - person PCR reaction system is shown in Table 2.

[0080] Table 2 Single - person PCR reaction system

[0081] Component Volume Primer-probe mixture 6 μL Premix 11 μL

[0082] Step S30: Add the nucleic acid of the test sample, CT positive control, and CT negative control to the PCR reaction tubes respectively, and transfer them to the droplet preparation area after instantaneous centrifugation.

[0083] Taking the above single - person PCR reaction system as an example, add 5 μL of the nucleic acid of the test sample, CT positive control, and CT negative control to the PCR reaction tubes after adding the PCR reaction system respectively. Tighten the tube caps, and transfer them to the droplet preparation area after instantaneous centrifugation for 15 seconds.

[0084] Step S40: Use a droplet preparation chip to prepare droplets from the liquid in the PCR reaction tube, and perform ddPCR amplification reaction on the prepared droplets according to the PCR amplification conditions to obtain amplification products.

[0085] Prepare droplets according to the instruction manual of Bio-rad's droplet digital PCR platform, and transfer the prepared droplets to a dedicated 96-well plate for Bio-rad's droplet digital PCR.

[0086] Specifically, mix the PCR reaction system with the added sample using a vortex oscillator. Take out a droplet preparation chip, transfer a column (8) of the reaction system to the sample wells of the chip, add 70 μL of droplet preparation oil to the droplet preparation oil wells, and send it into the droplet preparation instrument for droplet preparation. After the instrument reports that the droplet preparation is completed, carefully transfer the prepared droplets to a dedicated 96-well plate using a pipette, discard the droplet preparation chip, and then take another droplet preparation chip to process the next column of the reaction system until all the reaction systems to be tested are processed. Then seal the 96-well plate with an aluminum mold on a heat sealer.

[0087] Transfer the sealed 96-well plate to a qualitative PCR, and perform a PCR reaction according to the PCR amplification conditions set in Table 3. Among them, the PCR amplification conditions are: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing and extension at 58°C for 1 min, for a total of 45 cycles; then, enzyme inactivation at 98°C for 10 min; and finally, hold at 4°C.

[0088] Table 3 PCR amplification conditions

[0089]

[0090]

[0091] After PCR is completed, take out the 96-well plate containing the amplification products and transfer it to a droplet reader.

[0092] Step S50: Perform quantitative analysis on the amplification products to obtain the copy number of Chlamydia trachomatis nucleic acid in the sample to be tested.

[0093] Set the droplet reading parameters according to the instruction manual, pay attention to the fluorescence channel selection: select the FAM channel to detect Chlamydia trachomatis nucleic acid, select the VIC channel to detect the internal standard (internal control gene), and after setting is completed, start the droplet reading program.

[0094] After the droplet reading is completed, the results are automatically saved and enter the result analysis interface. Check the total number of droplets in each reaction well. If the number of droplets in a certain reaction well < 10000, it is regarded as an invalid reaction well. Then check whether the positive and negative quality control products of this test are included in the valid reaction wells. If they are included, this test is valid.

[0095] See Figure 1 and Figure 2 the PCR reaction results of the kit of the present application for detecting CT negative control product and CT positive control product shown in the figure. Among them, Ch1 corresponds to the FAM fluorescence channel, and Ch2 corresponds to the VIC fluorescence channel. As can be seen from the figure, in Figure 1 , no signal is detected through the Ch1 channel, and the internal control gene fragment is detected through the Ch2 channel; Figure 2 , the Chlamydia trachomatis gene fragment and the internal control gene fragment are detected through the Ch1 channel and the Ch2 channel respectively.

[0096] If the test is valid, view the fluorescence scatter plot of Ch1 (FAM channel). After drawing the threshold line, view the concentration of Ch1, in units of copies / μL. Finally, the concentration of the original sample can be calculated by combining the sample dilution factor.

[0097] It should be noted that the detection principle of the present application is as follows:

[0098] The droplet digital PCR technology of Bio-rad in this kit is used to design specific primers and probes for the Chlamydia trachomatis OmpA gene, and Chlamydia trachomatis is labeled with FAM fluorescence, and the internal standard gene is labeled with VIC fluorescence. After preparing the PCR reaction system and adding samples, it is dispersed into tens of thousands of droplets, and each droplet can simultaneously perform PCR reactions. After the PCR reaction is completed, a droplet reader is used to detect the fluorescence level of each droplet to judge the positivity or negativity of the droplet. After counting the positivity or negativity of all droplets, it can be judged whether there is Chlamydia trachomatis nucleic acid in the sample. Further, according to the statistical results of the positive and negative droplets and combined with the statistical formula, the copy number of Chlamydia trachomatis nucleic acid in the sample can be calculated.

[0099] The following will further illustrate the content of the present application in more detail with specific examples and elaborate on the present application. However, these examples are by no means limiting the present application.

[0100] Example 1

[0101] This example provides the composition, packaging and quantity (96 person-times / box) of a kit for quantitatively detecting Chlamydia trachomatis nucleic acid, as shown in Table 4.

[0102] Table 4 Composition, packaging and quantity of a kit for quantitatively detecting Chlamydia trachomatis nucleic acid

[0103]

[0104] Example 2

[0105] This example conducts a detection experiment on the sample detection range and sensitivity of the kit of the present application. The specific process is as follows:

[0106] Take an appropriate amount of CT primer-probe mixture and ddPCR Supermix for Probes (No dUTP), prepare the PCR reaction system in the manner of step S20, and transfer it into a PCR reaction tube; select 1.19×10 7 copies / mL Chlamydia trachomatis culture, dilute it with a negative sample for 1:10 1 , 1:10 2 , 1:10 3 , 1:10 4 and 1:10 5 dilution. After dilution, the sample numbers are S1 to S5. After nucleic acid extraction, take 5 μL of the extraction solution and add it to the eight-well tube of the PCR reaction system prepared in step S20 to make the total volume 22 μL. Tighten the eight-well tube caps, mix vigorously with an oscillator for 15 seconds, centrifuge instantaneously for 15 seconds, and then transfer to the droplet preparation area.

[0107] After preparing the droplets according to the droplet preparation method of step S40 above, seal the 96-well plate with an aluminum mold on a heat sealer, transfer the sealed 96-well plate to a qualitative PCR, and perform a PCR reaction on the prepared droplets according to the PCR amplification conditions in Table 3.

[0108] After the reaction ends, the reaction results are shown in Table 5 and Figures 3 to 7 , Figures 3 to 7 in turn are the PCR reaction results (i.e., amplification products) of S1 to S5 with 1:10 1 , 1:10 2 , 1:10 3 , 1:10 4 and 1:10 5 .

[0109] Table 5 Detection results of sample sensitivity

[0110]

[0111] As can be seen from the above table results, when the sample concentration is greater than 1000 copies / mL, the quantitative test result of the kit of the present application has a difference of <15% from the true concentration. The results show that the lowest detection limit of the kit of the present application is 1000 copies / mL, and the kit has high sensitivity.

[0112] Example 3

[0113] In this embodiment, the accuracy of the kit of the present application was detected. Specifically, the kit of the present application was used to detect positive reference products (PC01-PC10) and negative reference products (including high-risk human papillomavirus type 16, high-risk human papillomavirus type 18, herpes simplex virus type II, Candida albicans, Serratia marcescens, Trichomonas vaginalis, adenovirus, cytomegalovirus, group B streptococcus) (NC01-NC9). Take 5 μL of the extraction solution and add it to the eight-well strip tube of the PCR reaction system prepared in step S20 to make the total volume 22 μL. Tighten the lids of the eight tubes, mix well by vigorous oscillation with an oscillator for 15 seconds, and transfer to the droplet preparation area after instantaneous centrifugation for 15 seconds.

[0114] After the droplets were prepared according to the droplet preparation method in step S40 above, the 96-well plate was sealed with an aluminum mold on a heat sealer, and the sealed 96-well plate was transferred to a qualitative PCR. The prepared droplets were subjected to PCR reaction according to the PCR amplification conditions in Table 3.

[0115] After the reaction ended, the reaction results are shown in Table 6 and Figure 8 、 Figure 9 。

[0116] Table 6 Accuracy test results

[0117]

[0118]

[0119] The test results showed that the coincidence rate of the negative reference products was 100%, the coincidence rate of the positive reference products was 100%, and the negative-positive coincidence rate of the accuracy test results of each reference product was 100%, indicating that the accuracy test of the kit of the present application met the requirements, further indicating high detection accuracy and accurate quantitative results.

[0120] Example 4

[0121] This embodiment was an application experiment. The kit of the present application and a third-party chlamydia trachomatis detection kit were used to detect the positive and negative of the test samples.

[0122] Ten male urethral chlamydia trachomatis positive samples, ten male urethral chlamydia trachomatis negative samples, ten female genital tract chlamydia trachomatis positive samples, and ten female genital tract chlamydia trachomatis negative samples were respectively selected. After nucleic acid extraction, the samples were labeled and the label information was ensured to be correct, and stored at -80 °C. During the experiment, 5 μL of each sample was taken and added to the reaction tube containing the PCR reaction solution prepared according to step S20 to make the total volume 22 μL. Mix well by vigorous oscillation with an oscillator for 15 seconds, and transfer to the droplet preparation area after instantaneous centrifugation for 15 seconds.

[0123] After the microdroplets are prepared by the microdroplet preparation method in step S40 above, seal the 96-well plate with an aluminum mold on a heat sealer, transfer the sealed 96-well plate to a qualitative PCR, and perform a PCR reaction on the prepared microdroplets according to the PCR amplification conditions in Table 3.

[0124] After the reaction is completed, the reaction results are shown in Table 7 and Figures 10 to 13 as shown.

[0125] Table 7 Sample detection results of the kit of the present application and third-party kits

[0126]

[0127]

[0128]

[0129] The detection results show that among 40 samples, 20 are positive for Chlamydia trachomatis and 20 are negative. The consistency of the detected results with those of the third-party kit is 100%. Through the detection of the kit of the present application, the qualitative and quantitative detection of suspected Chlamydia trachomatis nucleic acid fragments is worthy of popularization and application.

[0130] Obviously, the embodiments described above are only a part of the embodiments of the present application, rather than all of them. The preferred embodiments of the present application are given in the drawings, but do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions described in the foregoing specific embodiments, or perform equivalent replacements on some of the technical features. Any equivalent structure made by using the specification and drawings of the present application, directly or indirectly applied in other related technical fields, is equally within the scope of the patent protection of the present application.

Claims

1. A primer and a probe for quantitatively detecting Chlamydia trachomatis nucleic acid, characterized in that, The primers include an upstream primer for detecting Chlamydia trachomatis, a downstream primer for detecting Chlamydia trachomatis, an upstream primer for detecting an internal control gene, and a downstream primer for detecting an internal control gene; the probes include a probe for detecting Chlamydia trachomatis and an internal control probe; Among them, the nucleotide sequence of the upstream primer for detecting Chlamydia trachomatis is shown as SEQ ID NO: 1, and the nucleotide sequence of the downstream primer for detecting Chlamydia trachomatis is shown as SEQ ID NO: 2; the nucleotide sequence of the upstream primer for detecting the internal control gene is shown as SEQ ID NO: 3, and the nucleotide sequence of the downstream primer for detecting the internal control gene is shown as SEQ ID NO: 4; The nucleotide sequence of the probe for detecting Chlamydia trachomatis is shown as SEQ ID NO: 5, and the nucleotide sequence of the internal control probe is shown as SEQ ID NO:

6.

2. The primer and probe for quantitatively detecting Chlamydia trachomatis nucleic acid according to claim 1, characterized in that, The 5' end of the nucleotide sequences corresponding to the probe for detecting Chlamydia trachomatis and the internal control probe is labeled with a fluorescent group; the 3' end of the nucleotide sequences corresponding to the probe for detecting Chlamydia trachomatis and the internal control probe is labeled with a quenching group; Among them, the fluorescent group is selected from FAM, HEX or VIC; the quenching group is selected from MGB, BHQ, TAMRA or Eclipse.

3. A kit for quantitatively detecting Chlamydia trachomatis nucleic acid, characterized in that, It includes a primer-probe mixture, a premix, a CT positive control product and a CT negative control product; among them, the primer-probe mixture includes the primers and probes for quantitatively detecting Chlamydia trachomatis nucleic acid as described in claim 1 or 2.

4. The kit for quantitatively detecting Chlamydia trachomatis nucleic acid according to claim 3, characterized in that, The final concentrations of the upstream primer for detecting Chlamydia trachomatis and the downstream primer for detecting Chlamydia trachomatis in the PCR reaction system are 0.50 - 0.70 μmol / L respectively; the final concentration of the probe for detecting Chlamydia trachomatis in the PCR reaction system is 0.20 - 0.30 μmol / L; The final concentrations of the upstream primer for detecting the internal control gene and the downstream primer for detecting the internal control gene in the PCR reaction system are 0.40 - 0.60 μmol / L respectively; the final concentration of the internal control probe in the PCR reaction system is 0.20 - 0.30 μmol / L.

5. The kit for quantitatively detecting Chlamydia trachomatis nucleic acid according to claim 3, wherein The components of the premix include Tris-HCl buffer, dNTP, and PCR reaction enzyme.

6. The kit for quantitatively detecting Chlamydia trachomatis nucleic acid according to claim 3, wherein, The components of the CT positive control product include inactivated Chlamydia trachomatis culture and pseudovirus containing an internal standard fragment, and the components of the CT negative control product include pseudovirus containing an internal standard fragment and TE buffer.

7. A detection method for quantitatively detecting Chlamydia trachomatis nucleic acid, which uses the kit described in any one of claims 3 to 6 to detect Chlamydia trachomatis nucleic acid for non-diagnostic purposes, characterized in that, It includes the following steps: Collect a sample to be tested for Chlamydia trachomatis, extract nucleic acid from the sample to be tested to obtain the nucleic acid of the sample to be tested; Take out the primer-probe mixture and the premix from the kit, melt them at room temperature and mix well to prepare a PCR reaction solution system, and dispense the PCR reaction solution system into a preset number of PCR reaction tubes; Correspondingly add the nucleic acid of the sample to be tested, the CT positive control product and the CT negative control product into the PCR reaction tubes, centrifuge instantaneously and transfer to the droplet preparation area; Use a droplet preparation chip to prepare droplets for the liquid in the PCR reaction tubes, and perform ddPCR amplification reaction on the prepared droplets according to the PCR amplification conditions to obtain amplification products; Quantitative analysis is performed on the amplified product to obtain the copy number of Chlamydia trachomatis nucleic acid in the sample to be tested.

8. The detection method for quantitatively detecting Chlamydia trachomatis nucleic acid according to claim 7, wherein, The sample to be tested for Chlamydia trachomatis uses a human urogenital system sample.

9. The detection method for quantitatively detecting Chlamydia trachomatis nucleic acid according to claim 7, characterized in that, The detection method further includes: Take the Chlamydia trachomatis sample to be tested with a concentration of (1.15 - 1.20)×10 7 copies / mL, and use the negative sample to dilute the sample to be tested at ratios of 1:10, 1:10 2 , 1:10 3 , 1:10 4 , and 1:10 5 to obtain samples to be tested with different dilution concentrations, numbered S1 - S5; Nucleic acid extraction is performed on the samples to be tested S1 to S5 with different dilution concentrations to obtain the nucleic acids of the samples to be tested corresponding to different dilution concentrations.

10. The method for detecting Chlamydia trachomatis according to claim 7, characterized in that, The PCR amplification conditions are as follows: Pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing and extension at 58°C for 1 min, for a total of 45 cycles; Then, enzyme inactivation at 98°C for 10 min; finally, hold at 4°C.

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