Multiple primer group for detecting reproductive system disease pathogens and kit thereof

Abstract

The invention discloses a multiple primer group for detecting pathogens of reproductive system diseases and a kit thereof. The kit comprises a combination of amplification primers and fluorescent probes of 10 pathogens, the pathogens comprise molluscum contagiosum virus, Snethia bacteria, gardnerella vaginalis, prevotella genus, campylobacter genus, giant globules genus, Atortobacter vaginalis, lactobacillus jensenii, lactobacillus crispatus and inert lactobacillus. The kit contains UDG enzyme and is used for preventing pollution of a PCR amplification product, and the accuracy of a detection result is ensured. The kit provided by the invention contains an internal reference control, can display the occurrence of false negative, and is used for monitoring the collection, transportation, extraction and amplification processes of a to-be-detected sample. According to the kit, the specificity is 100%, the detection sensitivity reaches 10 copies / mL, the specificity is good, and the detection efficiency is high. The kit is used for identifying the reproductive system disease pathogens of a to-be-detected sample, the to-be-detected sample can be vaginal secretion, the operation steps are simple, and the detection period is short.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a multiple primer set and a kit thereof for detecting pathogens of reproductive system diseases. Background Art

[0002] In recent years, the number of patients with urogenital tract infections has continued to increase. Common epidemiological surveys of married women show that the prevalence of such infections has become the highest among gynecological diseases. The pathogens that cause genital tract infections are numerous and complex, and there are certain asymptomatic infections, which cause many difficulties in the diagnosis and treatment of the disease. Reproductive tract pathogen infections usually lead to the following symptoms: urinary tract infections, vaginitis, pelvic inflammatory disease, etc. Reproductive tract pathogen infections will increase the incidence of male and female infertility, ectopic pregnancy, miscarriage, premature birth and other symptoms in pregnant women; infections such as gonorrhea and syphilis can be transmitted to children, affecting the physical fitness of newborns and the health of the next generation; chronic inflammation of the reproductive system will increase the chance of HIV infection.

[0003] Reproductive tract infection can cause damage to human reproductive function and is an important cause of infertility in many patients. 20%-60% of female infertility is caused by infection with reproductive tract pathogens. Vaginitis is an extremely common gynecological disease, ranking first among women's reproductive tract infections. Its occurrence and development are often the result of the combined action of multiple factors. Reproductive tract pathogens are complex and diverse, including bacteria, fungi, Trichomonas, Mycoplasma, Chlamydia, Neisseria gonorrhoeae, viruses, etc. The reproductive tract infections caused by them are closely related to the occurrence and development of vaginitis. Sexually transmitted diseases caused by Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, papillomavirus, etc. can also affect the normal function of organs and induce infertility.

[0004] Clinically, the pathogens that cause reproductive tract infections are complex and diverse, and difficult to diagnose. There is an urgent need to seek a highly specific, highly sensitive, rapid, and economical detection method for multiple pathogens to provide an experimental basis for the diagnosis and treatment of clinical pathogens. At present, the main detection methods for pathogens of reproductive tract infections in clinical practice include pathogen culture, immunoassay, and fluorescence quantitative PCR. Virus culture detection has the characteristics of high specificity and is the "gold standard" for clinical diagnosis, but culture is time-consuming, low in sensitivity, and requires high experimental operation; bacterial culture can detect drug resistance, but samples are easily contaminated, which has a great impact on the results; immunological detection of pathogens of reproductive tract infections often uses enzyme-linked immunosorbent assay, which has the advantages of being intuitive, mature in technology, and good in specificity, but it is time-consuming, cumbersome in operation, low in sensitivity, and high in false positives, which limits its reference value in clinical practice; fluorescence quantitative PCR technology has the advantages of high sensitivity, rapid detection, and relatively low cost, and has been widely used in clinical diagnosis. The existing multiplex PCR kit has the problem that as the number of detection increases, the possibility of primer dimer formation increases, which may lead to nonspecific amplification, reduce the target amplification efficiency, and affect the detection sensitivity and specificity. Although patented technologies for primer sets and kits for multiplex PCR detection of the reproductive system are available, the existing technology has not yet provided a rapid, accurate and easy-to-operate multiplex PCR detection method for detecting individual pathogens in the reproductive system.

[0005] The use of the pathogen markers and primers thereof provided by the present invention in the preparation of products for diagnosing reproductive tract infections can alleviate the technical problem that the prior art still has insufficient research on the diagnosis of specific pathogens related to reproductive tract infections, such as molluscum contagiosum virus and Sneathia bacteria. Usually, multiplex PCR detection uses 4 channels in the same tube, that is, 4 pathogens are detected. The present invention can detect 6 pathogens in the same tube. Summary of the invention

[0006] The present invention aims to solve the problems of difficulty in missing individual pathogens and complex screening in existing pathogen detection technologies related to reproductive system diseases, and provides a new multiple primer set and kit for detecting pathogens of reproductive system diseases. The pathogens include Molluscum contagiosum virus (MCV), Sneathia (SB), Gardnerella vaginalis (GV), Prevotella (PB), Campylobacter (CB), Megasphaera (MB), Atopobium vaginae (AV), Lactobacillus jensenii (LJ), Lactobacillus crispatus (LC), and Lactobacillus iners (LI).

[0007] Specifically, the technical solution provided by this application is as follows: In a first aspect, the present invention provides a multiplex primer set for detecting 10 pathogens of reproductive system diseases, comprising: The forward primer for detecting molluscum contagiosum virus is shown in SEQ ID NO. 1, the reverse primer is shown in SEQ ID NO. 2, and the fluorescent probe is shown in SEQ ID NO. 3; The forward primer for detecting Sneathia bacteria is shown in SEQ ID NO. 4, the reverse primer is shown in SEQ ID NO. 5, and the fluorescent probe is shown in SEQ ID NO. 6; The forward primer for detecting Gardnerella vaginalis is shown in SEQ ID NO.7, the reverse primer is shown in SEQ ID NO.8, and the fluorescent probe is shown in SEQ ID NO.9; The forward primer for detecting Prevotella is shown in SEQ ID NO. 10, the reverse primer is shown in SEQ ID NO. 11, and the fluorescent probe is shown in SEQ ID NO. 12; The forward primer for detecting Campylobacter is shown in SEQ ID NO. 13, the reverse primer is shown in SEQ ID NO. 14, and the fluorescent probe is shown in SEQ ID NO. 15; The forward primer for detecting Megasphaera is shown in SEQ ID NO. 16, the reverse primer is shown in SEQ ID NO. 17, and the fluorescent probe is shown in SEQ ID NO. 18; The forward primer for detecting Atopobium vaginalis is shown in SEQ ID NO. 19, the reverse primer is shown in SEQ ID NO. 20, and the fluorescent probe is shown in SEQ ID NO. 21; The forward primer for detecting Lactobacillus jensenii is shown in SEQ ID NO. 22, the reverse primer is shown in SEQ ID NO. 23, and the fluorescent probe is shown in SEQ ID NO. 24; The forward primer for detecting Lactobacillus crispatus is shown in SEQ ID NO. 25, the reverse primer is shown in SEQ ID NO. 26, and the fluorescent probe is shown in SEQ ID NO. 27; The forward primer for detecting Lactobacillus inerans is shown in SEQ ID NO. 28, the reverse primer is shown in SEQ ID NO. 29, and the fluorescent probe is shown in SEQ ID NO. 30.

[0008] Based on the above technical solution, the 5' end of the fluorescent probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

[0009] Based on the above technical solution, the fluorescent group is selected from any one of Atto 425, FAM, ROX, CY5, CY5.5 and VIC, and the quenching group is any one of BHQ1, BHQ2 and BHQ3.

[0010] In a second aspect, based on the same invention, the present invention also provides a kit for detecting pathogens of reproductive system diseases, wherein the kit comprises a multiple primer set for detecting 10 pathogens of reproductive system diseases as described in the first aspect.

[0011] Based on the above technical solution, the kit comprises reaction solution A, reaction solution B, enzyme mixture, positive control substance and negative control substance.

[0012] Further, the reaction solution A includes a reaction premix, a molluscum contagiosum virus forward primer, a molluscum contagiosum virus reverse primer, a molluscum contagiosum virus fluorescent probe, a Sneathia bacteria forward primer, a Sneathia bacteria reverse primer, a Sneathia bacteria fluorescent probe, a Gardnerella vaginalis forward primer, a Gardnerella vaginalis reverse primer, a Gardnerella vaginalis fluorescent probe, a Prevotella forward primer, a Prevotella reverse primer, a Prevotella fluorescent probe, a Campylobacter forward primer, a Campylobacter reverse primer, a Campylobacter fluorescent probe, a human ribonuclease forward primer, a human ribonuclease reverse primer, and a human ribonuclease fluorescent probe.

[0013] Further, the reaction solution B includes a reaction premix, a Megasphaera forward primer, a Megasphaera reverse primer, a Megasphaera fluorescent probe, a vaginal Atopobium forward primer, a vaginal Atopobium reverse primer, a vaginal Atopobium fluorescent probe, a Lactobacillus jensenii forward primer, a Lactobacillus jensenii reverse primer, a Lactobacillus jensenii fluorescent probe, a Lactobacillus crispatus forward primer, a Lactobacillus crispatus reverse primer, a Lactobacillus crispatus fluorescent probe, a Lactobacillus iners forward primer, a Lactobacillus iners reverse primer, a Lactobacillus iners fluorescent probe, a human ribonuclease forward primer, a human ribonuclease reverse primer, and a human ribonuclease fluorescent probe.

[0014] Furthermore, the forward primer of the human ribonuclease (RP) is shown in SEQ ID NO. 31, the reverse primer is shown in SEQ ID NO. 32, and the fluorescent probe is shown in SEQ ID NO. 33; Furthermore, the reaction premix includes 1×PCR reaction buffer and RNase free H2O.

[0015] Furthermore, the enzyme mixture includes 1U / μL Taq DNA polymerase and 0.2U / μL UDG enzyme.

[0016] 10. A kit for detecting pathogens of reproductive system diseases according to claim 5, characterized in that the positive control substance comprises 10 pathogens and RP gene sequences; and the negative control substance is TE Buffer.

[0017] Furthermore, in reaction solution A, the fluorescent groups of the six fluorescent probes, namely, molluscum contagiosum virus (MCV), Sneathia bacteria (SB), Gardnerella vaginalis (GV), Prevotella (PB), Campylobacter (CB) and human ribonuclease RNase fragment, are different; in reaction solution B, the fluorescent groups of the six fluorescent probes, namely, Megasphaera (MB), Atopobium vaginalis (AV), Lactobacillus jensenii (LJ), Lactobacillus crispatus (LC), Lactobacillus iners (LI) and human ribonuclease RNase fragment, are different; but the fluorescent groups in reaction solution A and reaction solution B can be the same.

[0018] Further preferably, in reaction solution A, the 5' end of the fluorescent probe of MCV is labeled with Atto 425 fluorescent group, and the 3' end is labeled with quencher group BHQ1; the 5' end of the fluorescent probe of SB is labeled with FAM fluorescent group, and the 3' end is labeled with quencher group BHQ1; the 5' end of the fluorescent probe of GV is labeled with ROX fluorescent group, and the 3' end is labeled with quencher group BHQ2; the 5' end of the fluorescent probe of PB is labeled with CY5 fluorescent group, and the 3' end is labeled with quencher group BHQ3; the 5' end of the fluorescent probe of CB is labeled with CY5.5 fluorescent group, and the 3' end is labeled with quencher group BHQ3; the 5' end of the fluorescent probe of RP is labeled with VIC fluorescent group, and the 3' end is labeled with quencher group BHQ1; Further preferably, in reaction solution B, the 5' end of the fluorescent probe of MB is labeled with an Atto 425 fluorescent group, and the 3' end is labeled with a quencher group BHQ1; the 5' end of the fluorescent probe of AV is labeled with a FAM fluorescent group, and the 3' end is labeled with a quencher group BHQ1; the 5' end of the fluorescent probe of LJ is labeled with a ROX fluorescent group, and the 3' end is labeled with a quencher group BHQ2; the 5' end of the fluorescent probe of LC is labeled with a CY5 fluorescent group, and the 3' end is labeled with a quencher group BHQ3; the 5' end of the fluorescent probe of LI is labeled with a CY5.5 fluorescent group, and the 3' end is labeled with a quencher group BHQ3; the 5' end of the fluorescent probe of RP is labeled with a VIC fluorescent group, and the 3' end is labeled with a quencher group BHQ1.

[0019] The reaction principle of the kit is as follows: if during the qPCR reaction, if the sample to be tested contains MCV, the labeled Atto 425 probe in tube A containing reaction solution A generates a fluorescent signal; if the sample to be tested contains SB, the labeled FAM probe in tube A containing reaction solution A generates a fluorescent signal; if the sample to be tested contains GV, the labeled ROX probe in tube A containing reaction solution A generates a fluorescent signal; if the sample to be tested contains PB, the labeled CY5 probe in tube A containing reaction solution A generates a fluorescent signal; if the sample to be tested contains CB, the labeled CY5.5 probe in tube A containing reaction solution A generates a fluorescent signal; if the sample to be tested contains MB, the labeled Atto 425 probe generates a fluorescent signal; if the sample to be tested contains AV, the labeled FAM probe in tube B containing reaction solution B generates a fluorescent signal; if the sample to be tested contains LJ, the labeled ROX probe in tube B containing reaction solution B generates a fluorescent signal; if the sample to be tested contains LC, the labeled CY5 probe in tube B containing reaction solution B generates a fluorescent signal; if the sample to be tested contains LI, the labeled CY5.5 probe in tube B containing reaction solution B generates a fluorescent signal. In addition, if both tubes A and B contain internal reference genes, the labeled VIC probe should generate a fluorescent signal.

[0020] Furthermore, the specific primers and fluorescent probes of the present invention are shown in Table 1: Table 1: Primer and fluorescent probe sequence list

[0021] The present invention has the following advantages / benefits: The multiple primer set and kit for detecting pathogens of reproductive system diseases of the present invention can detect 10 pathogens simultaneously and specifically and sensitively. The multiple PCR kit for pathogens of reproductive system diseases includes amplification primers and fluorescent probe combinations of 10 parasites. Among them, the amplification reaction of 10 parasites contains multiple fluorescent probes, primers, enzyme mixtures, PCR reaction buffers, etc. of different pathogen targets, and can detect whether molluscum contagiosum virus (MCV), Sneathia bacteria (SB), Gardnerella vaginalis (GV), Prevotella (PB), Campylobacter (CB), Megasphaera (MB), Atopobium vaginalis (AV), Lactobacillus jensenii (LJ), Lactobacillus crispatus (LC) and Lactobacillus inertus (LI) in the same reaction system. The kit also includes positive controls and negative controls to monitor the accuracy of PCR detection.

[0022] The kit of this application contains UDG enzyme, which is used to prevent contamination of PCR amplification products and ensure the accuracy of the test results. The kit of this application contains an internal reference control, which can show the occurrence of false negatives and is used to monitor the collection, transportation, extraction and amplification process of the sample to be tested. The kit of this application has a specificity of 100% and a detection sensitivity of 10 copies / mL, with good specificity and high detection efficiency. The kit of this application is used to identify pathogens of reproductive system diseases in the sample to be tested. The sample to be tested can be vaginal secretions, with simple operation steps and short detection cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0024] Figure 1 : MCV test result diagram of the present invention; Figure 2 : SB detection result diagram of the present invention; Figure 3 : GV detection result diagram of the present invention; Figure 4 : PB test result diagram of the present invention; Figure 5: CB test result diagram of the present invention; Figure 6 : MB detection result diagram of the present invention; Figure 7 : AV detection result diagram of the present invention; Figure 8 : LJ detection result diagram of the present invention; Fig. 9 : LC detection result diagram of the present invention; Fig.10 : LI detection result diagram of the present invention; Fig.11 : The detection results of the five targets (MCV, SB, GV, PB, CB) and RP of the positive control of the present invention; Fig.12 : The detection results of the five targets (MB, AV, LJ, LC, LI) and RP of the positive control of the present invention; Fig.13 : 10 targets and RP detection results of the negative control of the present invention. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the present invention, unless otherwise specified, can be obtained from commercial channels. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0026] Embodiment 1: The kit for simultaneously detecting 10 pathogens of reproductive system diseases includes: reaction solution A, reaction solution B, enzyme mixture, positive control and negative control: Reaction solution A includes reaction premix, MCV forward primer, MCV reverse primer, MCV fluorescent probe, SB forward primer, SB reverse primer, SB fluorescent probe, GV forward primer, GV reverse primer, GV fluorescent probe, PB forward primer, PB reverse primer, PB fluorescent probe, CB forward primer, CB reverse primer, CB fluorescent probe; reaction solution B includes reaction premix, MB forward primer, MB reverse primer, MB fluorescent probe, AV forward primer, AV reverse primer, AV fluorescent probe, LJ forward primer, LJ reverse primer, LJ fluorescent probe, LC forward primer, LC reverse primer, LC fluorescent probe, LI forward primer, LI reverse primer, LI fluorescent probe; reaction solution A and reaction solution B also include RP forward primer, RP reverse primer, RP fluorescent probe, respectively.

[0027] The reaction premix includes 1×PCR reaction buffer and RNase free H 2 O, the concentration of each primer pair and fluorescent probe is 100-200nmol / L; the primer pair concentration can be set to 200 nmol / L, and the fluorescent probe concentration can be set to 100 nmol / L. The enzyme mixture includes 1U / μL Taq DNA polymerase, 0.2U / μL UDG enzyme and 1U / μL reverse transcriptase; the positive control contains 10 pathogens and RNase P gene sequences; the negative control is TE Buffer.

[0028] The presence of molluscum contagiosum virus (MCV), Sneathia bacteria (SB), Gardnerella vaginalis (GV), Prevotella (PB), Campylobacter (CB), Megasphaera (MB), Atopobium vaginalis (AV), Lactobacillus jensenii (LJ), Lactobacillus crispatus (LC) and Lactobacillus iners (LI) was detected in two tubes A and B in the same reaction system. MCV, SB, GV, PB and CB were labeled with Atto 425, FAM, ROX, Cy5 and Cy5.5 respectively in tube A, and Atto 425, FAM, ROX, Cy5 and Cy5.5 were labeled respectively in MB, AV, LJ, LC and LI in tube B. Reaction solution A was placed in A and reaction solution B was placed in B. Both were provided with internal reference, which was RNase P fragment labeled with VIC. The internal reference can show the occurrence of false negatives and is used to monitor the collection, transportation, extraction and amplification process of the sample to be tested.

[0029] The detection is carried out on a common qPCR detection platform. The steps are as follows: Step S1: Reagent preparation First, take all reagents out of the refrigerator and equilibrate to room temperature. The PCR reaction solution standard is: take n×9μL of reaction solution A / B and n×1μL of enzyme mixture into a 1.5 mL centrifuge tube. Vortex the centrifuge tube for a few seconds and centrifuge for a few seconds.

[0030] Aliquot the PCR reaction solution. Aliquot 10 μL of the PCR reaction solution into thin-walled PCR reaction tubes.

[0031] Step S2, nucleic acid extraction: nucleic acid extraction is performed simultaneously on the sample to be tested (the sample to be tested is a plasmid simulation corresponding to each target, as shown in Table 4) and the positive control and negative control. Specifically, the nucleic acid extraction or purification reagent of Qingdao Han Tang Biotechnology Co., Ltd. is used. The operation refers to the instructions for nucleic acid extraction or purification reagent of Qingdao Han Tang Biotechnology Co., Ltd.; Step S3, adding samples: take out the PCR reaction tube containing the PCR reaction solution in step S1, add 40 μL of the nucleic acid extracted in step S2, the positive control substance and the negative control substance nucleic acid, cover the tube tightly, shake evenly and then centrifuge instantly at a speed of 3000 rpm / min for 20 s; Step S4, amplification detection: placing the PCR reaction tube obtained in step S3 into a fluorescent PCR amplification instrument for amplification detection, and the reaction program is set as shown in Table 2; Table 2 Fluorescence amplification instrument reaction program

[0032] Step S5, result determination: determine the sample test result according to the determination criteria in Table 3.

[0033] Table 3 Judgment criteria

[0034] Molluscum contagiosum virus (MCV), Sneathia bacteria (SB), Gardnerella vaginalis (GV), Prevotella (PB), Campylobacter (CB), Megasphaera (MB), Atopobium vaginalis (AV), Lactobacillus jensenii (LJ), Lactobacillus crispatus (LC) and Lactobacillus iners (LI) as well as positive and negative controls were tested and analyzed. The positive and negative controls met the requirements, indicating that the experiment was effective. MCV, SB, GV, PB, CB, MB, AV, LJ, LC and LI were all detected on the corresponding targets, and no amplification was found in the negative controls, indicating that the detection kit and method have good specificity (the results are shown in Table 4).

[0035] Table 4 Sample test table

[0036] Example 2: Sensitivity detection The homemade 10 target standard products of 106 copies / mL were gradiently diluted to 103 copies / mL, 102 copies / mL, and 10 copies / mL as templates. The negative sample is normal body fluid. The diluted template and negative sample were amplified using the kit described in Example 1, respectively. The amplification conditions were the same as those in Example 1. The results of the 6 target detection sensitivity were determined according to the result judgment criteria in Example 1. Table 5 shows that when the target template is 10 copies / mL, the kit described in Example 1 can still use it as a template for amplification. Therefore, in the case of low template concentration, the kit of the present invention can still perform detection and thus has a very high sensitivity. The pathogen detection results with a sample concentration of 102 copies / mL are shown in Table 5. Figures 1 to 9 When the kit of the present invention is used, there is no cross reaction between the primer pairs, and the sensitivity is as high as that of ordinary qPCR.

[0037] Table 5 Detection sensitivity results of nucleic acid sample kits for 10 pathogens

[0038] Example 3: Specific detection Other common pathogens of reproductive system diseases including Neisseria gonorrhoeae, Treponema pallidum, herpes simplex virus, human immunodeficiency disease, Trichomonas vaginalis, Haemophilus vaginalis and Mycoplasma genitalium were selected as simulated interference samples, specifically homemade plasmids with a concentration of 106 copies / mL, for specific detection. The detection process is shown in Example 1. The results show that except for the positive control, the results of other samples are negative, proving that the present invention has no cross-reaction with other pathogen nucleic acids in this embodiment. The test results are shown in Table 6: Table 6: Sample test table

[0039] Example 4: Performance comparison of kits in different comparative examples This experiment provides kits of Comparative Example 1 and Comparative Example 2. The difference between these two kits and the kit of Example 1 is that different primer-probe compositions are used, as shown in Table 7. The fluorescent probes on each primer probe of each control group refer to Example 1 for comparative analysis, and the preparation method of the kit is the same as that of the aforementioned example.

[0040] The positive control substance in Example 1 was tested using the test kit prepared in Example 1 of the present application, and the test kits prepared in Control Group 1 and Control Group 2, respectively, to compare the experimental effects of each test kit.

[0041] Table 7 Primer probes for the control group

[0042] The experimental results show that compared with Example 1, the problems of Comparative Example 1 are: in Comparative Example 1, Atto 425 channels MCV and MD cannot be detected at 10 copies / mL; FAM channel SB cannot be detected at 10 copies / mL, and AV has a negative false positive; ROX channel GV cannot be detected at 10 copies / mL, and LJ has a negative false positive; CY5 channels PB and LC cannot be detected at 10 copies / mL; CY5.5 channel CB has a negative false positive, and LI cannot be detected at 10 copies / mL.

[0043] Compared with Example 1, the problem of Comparative Example 2 is that: in Comparative Example 1, Atto 425 channels MCV and MD cannot detect 10 copies / mL; FAM channel SB has a negative false positive, and AV cannot detect 10 copies / mL; ROX channels GV and LJ cannot detect 10 copies / mL; CY5 channel PB cannot detect 10 copies / mL, and LC has a negative false positive; CY5.5 channel CB cannot detect 10 copies / mL, and LI has a negative false positive.

[0044] In summary, the sensitivity and specificity of the multiple primer set and the detection kit thereof for detecting 10 types of lung abscess parasites provided by the present invention have significant effects.

[0045] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A multiplex primer set for detecting 10 pathogens of reproductive system diseases, characterized in that: include: The forward primer for detecting molluscum contagiosum virus is shown in SEQ ID NO. 1, the reverse primer is shown in SEQ ID NO. 2, and the fluorescent probe is shown in SEQ ID NO. 3; The forward primer for detecting Sneathia bacteria is shown in SEQ ID NO. 4, the reverse primer is shown in SEQ ID NO. 5, and the fluorescent probe is shown in SEQ ID NO. 6; The forward primer for detecting Gardnerella vaginalis is shown in SEQ ID NO.7, the reverse primer is shown in SEQ ID NO.8, and the fluorescent probe is shown in SEQ ID NO.9; The forward primer for detecting Prevotella is shown in SEQ ID NO. 10, the reverse primer is shown in SEQ ID NO. 11, and the fluorescent probe is shown in SEQ ID NO. 12; The forward primer for detecting Campylobacter is shown in SEQ ID NO. 13, the reverse primer is shown in SEQ ID NO. 14, and the fluorescent probe is shown in SEQ ID NO. 15; The forward primer for detecting Megasphaera is shown in SEQ ID NO. 16, the reverse primer is shown in SEQ ID NO. 17, and the fluorescent probe is shown in SEQ ID NO. 18; The forward primer for detecting Atopobium vaginalis is shown in SEQ ID NO. 19, the reverse primer is shown in SEQ ID NO. 20, and the fluorescent probe is shown in SEQ ID NO. 21; The forward primer for detecting Lactobacillus jensenii is shown in SEQ ID NO. 22, the reverse primer is shown in SEQ ID NO. 23, and the fluorescent probe is shown in SEQ ID NO. 24; The forward primer for detecting Lactobacillus crispatus is shown in SEQ ID NO. 25, the reverse primer is shown in SEQ ID NO. 26, and the fluorescent probe is shown in SEQ ID NO. 27; The forward primer for detecting Lactobacillus inerans is shown in SEQ ID NO. 28, the reverse primer is shown in SEQ ID NO. 29, and the fluorescent probe is shown in SEQ ID NO.

30.

2. A multiplex primer set for detecting 10 pathogens of reproductive system diseases according to claim 1, characterized in that: The 5' end of the fluorescent probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

3. A multiplex primer set for detecting 10 pathogens of reproductive system diseases according to claim 2, characterized in that: The fluorescent group is selected from any one of Atto 425, FAM, ROX, CY5, CY5.5 and VIC, and the quenching group is any one of BHQ1, BHQ2 and BHQ3.

4. A kit for detecting pathogens of reproductive system diseases, characterized in that: The kit comprises the multiple primer set for detecting 10 pathogens of reproductive system diseases as described in claim 1.

5. A kit for detecting pathogens of reproductive system diseases according to claim 4, characterized in that: The kit comprises a reaction solution A, a reaction solution B, an enzyme mixture, a positive control substance and a negative control substance.

6. A kit for detecting pathogens of reproductive system diseases according to claim 5, characterized in that: The reaction solution A includes a reaction premix, a molluscum contagiosum virus forward primer, a molluscum contagiosum virus reverse primer, a molluscum contagiosum virus fluorescent probe, a Sneathia bacteria forward primer, a Sneathia bacteria reverse primer, a Sneathia bacteria fluorescent probe, a Gardnerella vaginalis forward primer, a Gardnerella vaginalis reverse primer, a Gardnerella vaginalis fluorescent probe, a Prevotella forward primer, a Prevotella reverse primer, a Prevotella fluorescent probe, a Campylobacter forward primer, a Campylobacter reverse primer, a Campylobacter fluorescent probe, a human ribonuclease forward primer, a human ribonuclease reverse primer, and a human ribonuclease fluorescent probe.

7. A kit for detecting pathogens of reproductive system diseases according to claim 5, characterized in that: The reaction solution B includes a reaction premix, a Megasphaera forward primer, a Megasphaera reverse primer, a Megasphaera fluorescent probe, a vaginal Atopobium forward primer, a vaginal Atopobium reverse primer, a vaginal Atopobium fluorescent probe, a Lactobacillus jensenii forward primer, a Lactobacillus jensenii reverse primer, a Lactobacillus jensenii fluorescent probe, a Lactobacillus crispatus forward primer, a Lactobacillus crispatus reverse primer, a Lactobacillus crispatus fluorescent probe, a Lactobacillus iners forward primer, a Lactobacillus iners reverse primer, a Lactobacillus iners fluorescent probe, a human ribonuclease forward primer, a human ribonuclease reverse primer, and a human ribonuclease fluorescent probe.

8. A kit for detecting pathogens of reproductive system diseases according to any one of claims 6 or 7, characterized in that: The reaction premix includes 1×PCR reaction buffer and RNase free H2O.

9. A kit for detecting pathogens of reproductive system diseases according to claim 5, characterized in that: The enzyme mixture includes 1U / μL Taq DNA polymerase and 0.2U / μL UDG enzyme.

10. A kit for detecting pathogens of reproductive system diseases according to claim 5, characterized in that: The positive control contains 10 pathogens and RP gene sequences; the negative control is TE Buffer.

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