Primer probe combination, kit and method for quantitatively detecting herpes simplex virus type 2 nucleic acid

Abstract

The invention belongs to the technical field of biological detection, and relates to a primer and probe combination, a kit and a method for quantitatively detecting nucleic acid of herpes simplex virus type-2. The primer and the probe are characterized in that the nucleotide sequence of an upstream primer of the herpes simplex virus type-2 is shown as SEQ ID NO: 1, the nucleotide sequence of a downstream primer of the herpes simplex virus type-2 is shown as SEQ ID NO: 2, and the nucleotide sequence of the upstream primer of the herpes simplex virus type-2 is shown as SEQ ID NO: 2. The nucleotide sequence of the upstream primer of the internal control gene is as shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer of the internal control gene is as shown in SEQ ID NO: 4; the nucleotide sequence of the herpes simplex virus type 2 detection probe is as shown in SEQ ID NO: 5, and the nucleotide sequence of the internal control gene detection probe is as shown in SEQ ID NO: 6. According to the application, the sensitivity and accuracy of detecting the herpes simplex virus type 2 are improved.

Description

Technical Field

[0001] The present application relates to the field of biological detection technologies, and in particular, to a primer-probe combination, a kit, and a method for quantitatively detecting Herpes Simplex Virus Type 2 nucleic acid. Background Art

[0002] Herpes Simplex Virus (HSV) belongs to the genus Simplexvirus of the family Alphaherpesviridae. Common clinical manifestations of HSV infection are local herpes on mucous membranes or skin, and HSV can be divided into two serotypes, Type 1 (HSV-1) and Type 2 (HSV-2), according to antigenicity. Among them, HSV-1 infection is common in oral or facial herpes, while HSV-2 infection is the main cause (90%) of genital herpes. Genital herpes is a common ulcerative sexually transmitted disease, and its transmission route is direct contact or sexual contact. The virus generally initiates infection through damaged skin and mucous membranes.

[0003] HSV-2 infection mainly causes chronic, recurrent, and difficult-to-cure sexually transmitted diseases such as urogenital ulcers, cervical erosion, skin and mucous membrane damage in the perianal area, and neonatal herpes, and will increase the probability of syphilis and HIV infection, and will also synergistically induce cervical cancer with HPV. Women carrying HSV may transmit the virus to newborns during childbirth by contacting the infected site in the birth canal or through vaginal secretions containing HSV, resulting in neonatal HSV infection. 75% of neonatal herpes is caused by HSV-2.

[0004] However, most HSV-infected individuals have no typical symptoms or are asymptomatic, and the HSV vaccine is still in the early stage of research and development. Therefore, timely diagnosis and treatment measures for HSV infection have important clinical significance. Currently, for the detection of Herpes Simplex Virus Type 2, clinical applications mainly include culture methods, serological antibody detection, and nucleic acid detection, etc. Among them, the virus cultured by the culture method has low sensitivity and is not suitable for the accurate diagnosis of HSV-2; there are defects in using serological antibody detection to diagnose HSV-2 infection, which cannot truly reflect the infection situation and is prone to missed diagnosis or misdiagnosis; in addition, the above two methods and nucleic acid detection are all qualitative detections, which can only report the presence or absence of the target pathogen, and cannot meet the needs of quantitative detection. Summary of the Invention

[0005] The purpose of the embodiments of the present application is to propose a primer-probe combination, a kit, and a method for quantitatively detecting Herpes Simplex Virus Type 2 nucleic acid, so as to solve the technical problems that Herpes Simplex Virus Type 2 is difficult to quantitatively detect and the detection accuracy is not high.

[0006] To solve the above technical problems, an embodiment of the present application provides a primer-probe combination for quantitatively detecting Herpes simplex virus type 2 nucleic acid, adopting the following technical solution:

[0007] A primer-probe combination for quantitatively detecting Herpes simplex virus type 2 nucleic acid, comprising: primers and probes;

[0008] The primers include an upstream primer of Herpes simplex virus type 2, a downstream primer of Herpes simplex virus type 2, an upstream primer of an internal control gene, and a downstream primer of an internal control gene;

[0009] The nucleotide sequence of the upstream primer of Herpes simplex virus type 2 is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer of Herpes simplex virus type 2 is shown as SEQ ID NO: 2, the nucleotide sequence of the upstream primer of the internal control gene is shown as SEQ ID NO: 3, and the nucleotide sequence of the downstream primer of the internal control gene is shown as SEQ ID NO: 4;

[0010] The probes include a Herpes simplex virus type 2 detection probe and an internal control gene detection probe;

[0011] The nucleotide sequence of the Herpes simplex virus type 2 detection probe is shown as SEQ ID NO: 5, and the nucleotide sequence of the internal control gene detection probe is shown as SEQ ID NO: 6.

[0012] Further, the 5'-end of the Herpes simplex virus type 2 detection probe is labeled with a FAM fluorescent group, and the 5'-end of the internal control gene detection probe is labeled with a VIC fluorescent group; the 3'-ends of both the Herpes simplex virus type 2 detection probe and the internal control gene detection probe are labeled with an MGB quenching group.

[0013] To solve the above technical problems, an embodiment of the present application also provides a kit for quantitatively detecting Herpes simplex virus type 2 nucleic acid, adopting the following technical solution:

[0014] A kit for quantitatively detecting Herpes simplex virus type 2 nucleic acid, comprising:

[0015] A primer-probe mixture, a positive control product, a negative control product, and a droplet digital PCR premix;

[0016] The primer-probe mixture includes the above-mentioned primer-probe combination for quantitatively detecting Herpes simplex virus type 2 nucleic acid.

[0017] Further, the positive control product includes an inactivated culture of Herpes simplex virus type 2 and a pseudovirus containing an internal standard fragment.

[0018] Further, the negative control product includes TE and a pseudovirus containing an internal standard fragment.

[0019] Furthermore, the final concentration range of the upstream primer of herpes simplex virus type 2 in the PCR reaction system is 0.50 - 0.75 μmol / L, the final concentration range of the downstream primer of herpes simplex virus type 2 in the PCR reaction system is 0.50 - 0.75 μmol / L, the final concentration range of the detection probe for herpes simplex virus type 2 in the PCR reaction system is 0.20 - 0.30 μmol / L, the final concentration range of the upstream primer of the internal control gene in the PCR reaction system is 0.41 - 0.60 μmol / L, the final concentration range of the downstream primer of the internal control gene in the PCR reaction system is 0.41 - 0.60 μmol / L, and the final concentration range of the detection probe for the internal control gene in the PCR reaction system is 0.20 - 0.30 μmol / L.

[0020] To solve the above technical problems, the embodiments of the present application further provide a method for quantitatively detecting herpes simplex virus type 2 nucleic acid, adopting the following technical solutions:

[0021] A method for quantitatively detecting herpes simplex virus type 2 nucleic acid, which is detected by using the above-mentioned kit, is characterized by comprising the following steps:

[0022] S1. Collect genital and urinary tract secretions and herpes fluid swab samples;

[0023] S2. Extract the genital and urinary tract secretions and herpes fluid swab samples through a genital and urinary tract secretion and herpes fluid sample extraction kit to obtain the nucleic acid of the sample to be detected;

[0024] S3. Prepare a PCR reaction system and dispense the PCR reaction system into corresponding PCR reaction tubes;

[0025] S4. Add the nucleic acid of the sample to be detected, the negative control product, and the positive control product into the PCR reaction tubes respectively, tighten the tube caps, and perform instantaneous centrifugation;

[0026] S5. Prepare droplets according to the instructions of the Bio-rad droplet digital PCR platform, transfer the prepared droplets to a 96-well plate dedicated to Bio-rad droplet digital PCR, and thermally seal the 96-well plate with an aluminum film;

[0027] S6. Transfer the thermally sealed 96-well plate to a qualitative PCR instrument and perform PCR amplification according to the preset PCR reaction conditions to obtain an amplification result;

[0028] S7. Perform droplet reading and result validity analysis on the amplification result to obtain the copy number of herpes simplex virus type 2 nucleic acid.

[0029] Further, the PCR reaction system is prepared through the following steps:

[0030] Take out the primer-probe mixture and the droplet digital PCR premix, melt them at room temperature and mix them evenly by vortexing, and then centrifuge to obtain the PCR reaction system.

[0031] Further, the steps of the result validity analysis include:

[0032] Take the reaction wells with the number of droplets greater than or equal to 10,000 as valid reaction wells, and determine whether the positive control product or the negative control product exists in each of the valid reaction wells;

[0033] If the positive control product or the negative control product exists in each of the valid reaction wells, determine that the test result is valid, and obtain the fluorescence scatter plot and nucleic acid concentration in the FAM channel of the test result.

[0034] Further, the method further includes:

[0035] Use an inactivated culture of herpes simplex virus type 2 with a concentration range of 3×10 2 ~3×10 5 copies / mL as the sample to be tested;

[0036] Perform nucleic acid extraction, sample loading, droplet preparation, and PCR amplification on the sample to be tested, so that when the concentration range of the sample to be tested is 3×10 3 ~3×10 5 copies / mL, the difference between the sensitivity test result of the kit and the true concentration is less than or equal to 10%.

[0037] Compared with the prior art, the embodiments of the present application mainly have the following beneficial effects:

[0038] The primer-probe combination, kit, and method for quantitatively detecting herpes simplex virus type 2 nucleic acid provided by the present application optimize the specificity of the primers and probes, reduce the misdiagnosis probability caused by non-specific binding, and use the droplet digital PCR method to meet the need for quantitative detection of herpes simplex virus type 2 nucleic acid, improving the sensitivity and accuracy of detecting herpes simplex virus type 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] 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 drawings in the following description 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.

[0040] Figure 1 is a flowchart of an embodiment of a method for quantitatively detecting herpes simplex virus type 2 nucleic acid according to the present application;

[0041] Figure 2 is a PCR result diagram (FAM channel) of detecting a herpes simplex virus type 2 negative control product according to the present application;

[0042] Figure 3 is a PCR result diagram (VIC channel) of detecting a herpes simplex virus type 2 negative control product according to the present application;

[0043] Figure 4 is a PCR result diagram (FAM channel) of detecting a herpes simplex virus type 2 positive control product according to the present application;

[0044] Figure 5 is a PCR result diagram (VIC channel) of detecting a herpes simplex virus type 2 positive control product according to the present application;

[0045] Figure 6 is a sample detection result diagram (FAM channel) of Sample 1 in Example 2 according to the present application;

[0046] Figure 7 is a sample detection result diagram (FAM channel) of Sample 2 in Example 2 according to the present application;

[0047] Figure 8 is a sample detection result diagram (FAM channel) of Sample 3 in Example 2 according to the present application;

[0048] Figure 9 is a sample detection result diagram (FAM channel) of Sample 4 in Example 2 according to the present application;

[0049] Figure 10 is a sample detection result diagram (FAM channel) of Sample 5 in Example 2 according to the present application;

[0050] Figure 11 is a sample detection result diagram (FAM channel) of P1 in Example 3 according to the present application;

[0051] Figure 12 is a sample detection result diagram (FAM channel) of P2 in Example 3 according to the present application;

[0052] Figure 13 is a sample detection result diagram (FAM channel) of P3 in Example 3 according to the present application;

[0053] Figure 14 is a sample detection result diagram (FAM channel) of N1 in Example 3 according to the present application;

[0054] Figure 15 It is the sample detection result diagram (FAM channel) of N2 in Embodiment 3 of the present application;

[0055] Figure 16 It is the sample detection result diagram (FAM channel) of N3 in Embodiment 3 of the present application;

[0056] Figure 17 It is the sample detection result diagram (FAM channel) of N4 in Embodiment 3 of the present application;

[0057] Figure 18 It is the sample detection result diagram (FAM channel) of N5 in Embodiment 3 of the present application;

[0058] Figure 19 It is the sample detection result diagram (FAM channel) of N6 in Embodiment 3 of the present application;

[0059] Figure 20 It is the sample detection result diagram (FAM channel) of N7 in Embodiment 3 of the present application;

[0060] Figure 21 It is the detection situation diagram (FAM channel) of positive clinical samples numbered 1 - 8 in Embodiment 4 of the present application;

[0061] Figure 22 It is the detection situation diagram (FAM channel) of the positive clinical sample numbered 9 in Embodiment 4 of the present application;

[0062] Figure 23 It is the detection situation diagram (FAM channel) of positive clinical samples numbered 10 - 18 in Embodiment 4 of the present application;

[0063] Figure 24 It is the detection situation diagram (FAM channel) of clinical samples numbered 19 - 27 in Embodiment 4 of the present application;

[0064] Figure 25 It is the detection situation diagram (VIC channel) of negative clinical samples numbered 19 - 27 in Embodiment 4 of the present application;

[0065] Figure 26 It is the detection situation diagram (FAM channel) of clinical samples numbered 28 - 36 in Embodiment 4 of the present application;

[0066] Figure 27 It is the detection situation diagram (VIC channel) of negative clinical samples numbered 28 - 36 in Embodiment 4 of the present application. Detailed implementation manners

[0067] 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 "including" 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.

[0068] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in connection with the embodiments 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.

[0069] The following embodiments facilitate a better understanding of this application, but do not limit this application. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The experimental materials used in the following embodiments are all obtained from a conventional biochemical reagent store unless otherwise specified.

[0070] In order 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.

[0071] The embodiments of this application provide a primer-probe combination for quantitatively detecting herpes simplex virus type 2 nucleic acid, including: primers and probes;

[0072] The primers include an upstream primer of herpes simplex virus type 2, a downstream primer of herpes simplex virus type 2, an upstream primer of an internal control gene, and a downstream primer of an internal control gene;

[0073] The nucleotide sequence of the upstream primer of herpes simplex virus type 2 is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer of herpes simplex virus type 2 is shown as SEQ ID NO: 2, the nucleotide sequence of the upstream primer of the internal control gene is shown as SEQ ID NO: 3, and the nucleotide sequence of the downstream primer of the internal control gene is shown as SEQ ID NO: 4;

[0074] The probes include a herpes simplex virus type 2 detection probe and an internal control gene detection probe;

[0075] The nucleotide sequence of the herpes simplex virus type 2 detection probe is shown as SEQ ID NO: 5, and the nucleotide sequence of the internal control gene detection probe is shown as SEQ ID NO: 6.

[0076] In this embodiment, the nucleotide sequences are specifically as follows:

[0077] SEQ ID NO: 1 is 5'-TCCCCTGCTCTAGATATCCTC-3';

[0078] SEQ ID NO: 2 is 5'-CCGCGCCAAAGTTGT-3';

[0079] SEQ ID NO: 3 is 5'-AAATGAATGGGCAGCCGT-3';

[0080] SEQ ID NO: 4 is 5'-GAGAAATCGGGCCAGCTAG-3';

[0081] SEQ ID NO: 5 is 5'-CTGCCAAGGCGACCAGACAAAC-3';

[0082] SEQ ID NO: 6 is 5'-CTGCCGGTGACTAACCCTGCG-3'.

[0083] As an embodiment of the present application, the 5' end of the herpes simplex virus type 2 detection probe is labeled with a FAM fluorescent group, and the 5' end of the internal control gene detection probe is labeled with a VIC fluorescent group; the 3' ends of both the herpes simplex virus type 2 detection probe and the internal control gene detection probe are labeled with an MGB quenching group. Therefore, the 5' end of the nucleotide sequence of SEQ ID NO: 5 is labeled with a FAM fluorescent group, and the 5' end of the nucleotide sequence of SEQ ID NO: 6 is labeled with a VIC fluorescent group; the 3' ends of both the nucleotide sequence of SEQ ID NO: 5 and the nucleotide sequence of SEQ ID NO: 6 are labeled with an MGB quenching group.

[0084] Optionally, the length range of the primer is 15 - 30 bp, preferably 21 bp. This avoids non-specific amplification caused by too short primers and the reduction of the binding ability of primers to the target sequence due to too long primers; it further avoids the low PCR amplification efficiency caused by the inability of primers to fully bind to the target sequence. The length range of the final PCR amplification product is 80 - 250 bp (which can be extended to 300 bp).

[0085] Optionally, in the primer, the GC content is preferably 40%-60%, and the four bases are randomly distributed. Avoid the tandem arrangement of more than 5 purine or pyrimidine nucleotides, which may lead to the formation of secondary structures inside and result in poor amplification effects.

[0086] Optionally, the difference in Tm values of the primers is within 5°C, and the Tm value of the probe is at least 5°C higher than that of the primers.

[0087] Optionally, the fluorescent groups that the probe can be labeled with include FAM, HEX, JOE, TET, CY3, CY5, ROX, etc., and the quenching groups that the probe can be labeled with include MGB, BHQ, TAMRA, and Eclipse.

[0088] In this embodiment, the primer-probe combination of the present application, through specific design and selection of the US4 gene as the target gene, quantitatively detects herpes simplex virus type 2 nucleic acid on the basis of digital PCR technology. It should be noted that the US4 gene is located in the specific short region US of herpesvirus, and the envelope glycoprotein encoded by it can assist the virus in evading the host's clearance mechanism and participate in the infection of host cells by the virus; while the present application queries the US4 gene sequence of herpes simplex virus type 2 through the NCBI website, designs a primer-probe combination based on its specific conserved region, and then verifies the primer-probe specificity through the BLAST function, so as to determine the optimal primer-probe system and reaction procedure through repeated experiments to quantitatively detect herpes simplex virus type 2 nucleic acid.

[0089] The embodiment of the present application also provides a kit for quantitatively detecting herpes simplex virus type 2 nucleic acid, including: a primer-probe mixture, a positive control product, a negative control product, and a droplet digital PCR premix;

[0090] The primer-probe mixture includes the primer-probe combination for quantitatively detecting herpes simplex virus type 2 nucleic acid described above.

[0091] Among them, the droplet digital PCR premix can be expressed as ddPCR Supermix for Probes (NodUTP).

[0092] As an embodiment of the present application, the positive control product includes an inactivated culture of herpes simplex virus type 2 and a pseudovirus containing an internal standard fragment; among them, the concentration of the inactivated culture can be 3×10 5 copies / mL, and the internal standard concentration can be 5×10 5 copies / mL.

[0093] As an embodiment of the present application, the negative control product includes TE and pseudovirus containing an internal standard fragment; wherein, TE is 1×TE, and the internal standard concentration can be 1×10 5 copies / mL.

[0094] As an embodiment of the present application, the final concentration range of the upstream primer of herpes simplex virus type 2 in the PCR reaction system is 0.50 - 0.75 μmol / L, the final concentration range of the downstream primer of herpes simplex virus type 2 in the PCR reaction system is 0.50 - 0.75 μmol / L, the final concentration range of the detection probe of herpes simplex virus type 2 in the PCR reaction system is 0.20 - 0.30 μmol / L, the final concentration range of the upstream primer of the internal control gene in the PCR reaction system is 0.41 - 0.60 μmol / L, the final concentration range of the downstream primer of the internal control gene in the PCR reaction system is 0.41 - 0.60 μmol / L, and the final concentration range of the detection probe of the internal control gene in the PCR reaction system is 0.20 - 0.30 μmol / L;

[0095] Among them, the final concentration of the upstream primer of herpes simplex virus type 2 in the PCR reaction system is preferably 0.60 μmol / L, the final concentration of the downstream primer of herpes simplex virus type 2 in the PCR reaction system is preferably 0.60 μmol / L, the final concentration of the detection probe of herpes simplex virus type 2 in the PCR reaction system is preferably 0.25 μmol / L, the final concentration of the upstream primer of the internal control gene in the PCR reaction system is preferably 0.50 μmol / L, the final concentration of the downstream primer of the internal control gene in the PCR reaction system is preferably 0.50 μmol / L, and the final concentration of the detection probe of the internal control gene in the PCR reaction system is preferably 0.25 μmol / L.

[0096] The embodiment of the present application also provides a method for quantitatively detecting herpes simplex virus type 2 nucleic acid, which is detected using the above-mentioned kit, as Figure 1 shown, Figure 1 is a flowchart of an embodiment of the method for quantitatively detecting herpes simplex virus type 2 nucleic acid according to the present application;

[0097] This method includes the following steps:

[0098] Step S1, collect genital and urinary tract secretions and herpes fluid swab samples;

[0099] Step S2, extract the genital and urinary tract secretions and herpes fluid swab samples through a genital and urinary tract secretion and herpes fluid sample extraction kit to obtain the nucleic acid of the sample to be tested;

[0100] Step S3: Prepare the PCR reaction system and aliquot the PCR reaction system into corresponding PCR reaction tubes;

[0101] Step S4: Add the nucleic acid of the test sample, the negative control product, and the positive control product into the PCR reaction tubes respectively, tighten the tube caps, and perform instantaneous centrifugation;

[0102] Step S5: Prepare droplets according to the instructions of the Bio-rad droplet digital PCR platform, transfer the prepared droplets to a 96-well plate dedicated to Bio-rad droplet digital PCR, and heat-seal the 96-well plate with an aluminum film;

[0103] Step S6: Transfer the heat-sealed 96-well plate to a qualitative PCR instrument and perform PCR amplification according to the preset PCR reaction conditions to obtain an amplification result;

[0104] Step S7: Perform droplet reading and result validity analysis on the amplification result to obtain the copy number of herpes simplex virus type 2 nucleic acid.

[0105] As an embodiment of the present application, the PCR reaction system is prepared through the following steps:

[0106] Take out the primer-probe mixture and the droplet digital PCR premix, melt them at room temperature and mix them evenly by vortex oscillation, and centrifuge to obtain the PCR reaction system.

[0107] As an embodiment of the present application, the steps of the result validity analysis include:

[0108] Regard the reaction wells with the number of droplets greater than or equal to 10,000 as valid reaction wells, and determine whether the positive control product or the negative control product exists in each of the valid reaction wells;

[0109] If the positive control product or the negative control product exists in each of the valid reaction wells, determine that the test result is valid, and obtain the fluorescence scatter plot and nucleic acid concentration in the FAM channel of the test result.

[0110] As an embodiment of the present application, the method further includes:

[0111] Use an inactivated culture of herpes simplex virus type 2 with a concentration range of 3×10 2 ~3×10 5 copies / mL as the test sample;

[0112] Perform nucleic acid extraction, sample addition, droplet preparation, and PCR amplification on the test sample so that the concentration range of the test sample is 3×10 3 ~3×10 5When the concentration is [[ID=]], the difference between the sensitivity test result of the kit and the true concentration is less than or equal to 10%.

[0113] In this embodiment, the kit for quantitatively detecting Herpes simplex virus type 2 nucleic acid of the present application adopts the droplet digital PCR (i.e., "ddPCR") technology of Bio-rad to quantitatively detect Herpes simplex virus type 2 nucleic acid in the extracted reproductive and urinary tract secretions and herpes fluid swabs, which can reduce the misdiagnosis probability caused by non-specific binding and improve the sensitivity and accuracy of detecting Herpes simplex virus type 2.

[0114] The embodiment of the present application further illustrates the above scheme in combination with the method for quantitatively detecting Herpes simplex virus type 2 nucleic acid:

[0115] Step S1, sample collection:

[0116] Collect samples of reproductive and urinary tract secretions and herpes fluid swabs;

[0117] (1) Collection of female urethral / gential tract secretions (no urination for 2 hours before collecting urethral secretions): Wash the external secretions of the urethral orifice / cervix with a sterile saline cotton ball, then insert a sterile cotton swab about 2 cm into the urethra / cervix, twist the swab after 5 s to collect the secretion, place the cotton swab into a collection tube containing 1 mL of sterile saline, stir and wash thoroughly, squeeze the cotton swab dry, and cover the test tube stopper.

[0118] (2) Collection of male reproductive and urinary tract secretions (no urination for 2 hours before collecting secretions): Insert a small sterile cotton swab about 2 - 4 cm into the urethra, twist the swab after 5 s to collect the secretion, place the cotton swab into a glass test tube containing 1 mL of sterile saline, stir and wash thoroughly, squeeze the cotton swab dry, and cover the test tube stopper.

[0119] (3) Collection of herpes fluid:

[0120] Collection of blister fluid: Pierce the blisters on the external genitals of men and women, use a sterile cotton swab to collect the blister fluid and place it into a collection tube containing 1 mL of sterile saline, stir and wash thoroughly, squeeze the cotton swab dry, and cover the test tube stopper.

[0121] Collection of ulcer exudate: Remove the scab or dirt on the surface of the ulcer on the external genitals of men and women, use a sterile cotton swab to wipe or scrape the tissue fluid or exudate at the bottom of the ulcer or the unhealed part and place it into a glass test tube containing 1 mL of sterile saline, stir and wash thoroughly, squeeze the cotton swab dry, and cover the test tube stopper.

[0122] Step S2, sample extraction:

[0123] The reproductive and urinary tract secretion and herpes fluid swab samples are extracted by a reproductive and urinary tract secretion and herpes fluid sample extraction kit to obtain the nucleic acid of the sample to be tested.

[0124] Step S3: Prepare the PCR reaction system:

[0125] Prepare the PCR reaction system and dispense the PCR reaction system into corresponding PCR reaction tubes;

[0126] Take out the primer-probe mixture and the droplet digital PCR premix, melt them at room temperature and mix them evenly by vortexing, and centrifuge to obtain the PCR reaction system;

[0127] Specifically, take out the above primer-probe mixture and ddPCR Supermix for Probes (NodUTP) from the kit, melt and mix them evenly at room temperature, and centrifuge at 8000 rpm for several seconds to make all the liquid on the tube wall centrifuge to the bottom of the tube, so as to obtain the PCR reaction system. Then take N (N = the number of samples to be tested + HSV-2 negative control + HSV-2 positive control) PCR reaction tubes and dispense 17 μL of the PCR reaction system into the PCR reaction tubes.

[0128] Furthermore, the final concentration range of the upstream primer of herpes simplex virus type 2 in the PCR reaction system is 0.50 - 0.75 μmol / L, the final concentration range of the downstream primer of herpes simplex virus type 2 in the PCR reaction system is 0.50 - 0.75 μmol / L, the final concentration range of the detection probe of herpes simplex virus type 2 in the PCR reaction system is 0.20 - 0.30 μmol / L, the final concentration range of the upstream primer of the internal control gene in the PCR reaction system is 0.41 - 0.60 μmol / L, the final concentration range of the downstream primer of the internal control gene in the PCR reaction system is 0.41 - 0.60 μmol / L, and the final concentration range of the detection probe of the internal control gene in the PCR reaction system is 0.20 - 0.30 μmol / L;

[0129] Among them, the final concentration of the upstream primer of herpes simplex virus type 2 in the PCR reaction system is preferably 0.60 μmol / L, the final concentration of the downstream primer of herpes simplex virus type 2 in the PCR reaction system is preferably 0.60 μmol / L, the final concentration of the herpes simplex virus type 2 detection probe in the PCR reaction system is preferably 0.25 μmol / L, the final concentration of the upstream primer of the internal control gene in the PCR reaction system is preferably 0.50 μmol / L, the final concentration of the downstream primer of the internal control gene in the PCR reaction system is preferably 0.50 μmol / L, and the final concentration of the internal control gene detection probe in the PCR reaction system is preferably 0.25 μmol / L.

[0130] Step S4, sample addition:

[0131] Add the nucleic acid of the sample to be tested, the negative control product, and the positive control product into the PCR reaction tube respectively, tighten the tube cap, and perform instantaneous centrifugation;

[0132] Add 5 μL of the nucleic acid of the sample to be tested after extraction, the HSV-2 negative control product, and the HSV-2 positive control product into the above PCR reaction tube respectively, tighten the tube cap, and transfer it to the droplet preparation area after instantaneous centrifugation for 15 seconds.

[0133] Step S5, droplet preparation:

[0134] Perform droplet preparation according to the instructions of the droplet digital PCR platform of Bio-rad, transfer the prepared droplets to a 96-well plate dedicated to the droplet digital PCR of Bio-rad, and heat-seal the 96-well plate with an aluminum film;

[0135] 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 PCR reaction system to the sample wells of the chip, add 70 μL of droplet preparation oil to the droplet preparation oil wells, 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 PCR reaction system until all the PCR reaction systems to be tested are processed. Then seal the 96-well plate with an aluminum mold on the heat sealer.

[0136] Step S6, PCR amplification:

[0137] Transfer the heat-sealed 96-well plate to a qualitative PCR, and perform PCR amplification according to the preset PCR reaction conditions (see Table 1 below) to obtain the amplification result;

[0138] Table 1 PCR reaction conditions

[0139]

[0140] Step S7, Droplet Reading and Result Validity Analysis:

[0141] Perform droplet reading and result validity analysis on the amplification result to obtain the copy number of Herpes Simplex Virus Type 2 nucleic acid;

[0142] After the PCR is completed, transfer the 96-well plate to a droplet reader, set the droplet reading parameters according to the instruction manual, and pay attention to the fluorescence channel selection: select the FAM channel to detect Herpes Simplex Virus Type 2 nucleic acid, select the VIC channel to detect the internal standard, and start droplet reading after the settings are completed;

[0143] After the droplet reading is completed, the result is automatically saved. Click "Analyze" to enter the result analysis interface. First, click "Event" to view 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;

[0144] If the test is valid, click "1D Amplitude" to view the fluorescence scatter plot of Ch1. After drawing the threshold line, click "Concentration" to view the nucleic acid concentration of Ch1, with the unit of copies / μL. Combining the sample dilution factor, the nucleic acid concentration of the original sample can be calculated. As Figures 2 - 5 shown, where Figure 2 、 Figure 3 is the PCR result diagram of using the kit of the present application to detect the Herpes Simplex Virus Type 2 negative quality control product, Figure 2 is the FAM channel, Figure 3 is the VIC channel; Figure 4 、 Figure 5 is the PCR result diagram of using the kit of the present application to detect the Herpes Simplex Virus Type 2 positive quality control product, Figure 4 is the FAM channel, Figure 5 is the VIC channel.

[0145] The above scheme is further described below in conjunction with specific embodiments, but these embodiments are by no means a limitation to the present application. The preferred embodiments of the present application are described in detail as follows:

[0146] Example 1

[0147] Example 1 provides the composition, packaging, and quantity (96 person-times / box) of a kit for quantitatively detecting Herpes Simplex Virus Type 2 nucleic acid, as shown in Table 2 below:

[0148] Table 2 Composition, Packaging, and Quantity of the Kit

[0149]

[0150] The composition, packaging, and quantity of the herpes simplex virus type 2 nucleic acid quantitative detection kit provided in this embodiment can be specifically optimized for primers and probes, and an internal reference gene detection system is designed, reducing the misdiagnosis probability caused by non-specific binding.

[0151] Example 2

[0152] Example 2 provides a sample detection range and sensitivity detection experiment for a kit for quantitatively detecting herpes simplex virus type 2 nucleic acid.

[0153] Specifically, an appropriate number of copies of the HSV-2 primer-probe premix and ddPCR Supermix for Probes (NodUTP) are taken, prepared according to the method of step S3, and then transferred into a PCR reaction tube; 3×10 5 copies / mL, 3×10 4 copies / mL, 1.5×10 4 copies / mL, 3×10 3 copies / mL, 3×10 2 copies / mL of inactivated herpes simplex virus type 2 cultures are used as Sample1 to Sample5. After nucleic acid extraction, 5 μL of the extract is added to the octuplet PCR reaction system prepared in step S3 to make the total volume 22 μL. The octuplet tube lid is tightened, and it is vigorously shaken and mixed for 15 seconds with an oscillator, then centrifuged instantaneously for 15 seconds and transferred to the droplet preparation area.

[0154] The PCR reaction system with the added sample is mixed using a vortex oscillator. One droplet preparation chip is taken out, and a column (8) of the PCR reaction system is transferred to the sample wells of the chip. 70 μL of droplet preparation oil is added to the droplet preparation oil wells, and it is sent into the droplet preparation instrument for droplet preparation. After the instrument reports that the droplet preparation is completed, the prepared droplets are carefully transferred to a dedicated 96-well plate using a pipette. The droplet preparation chip is discarded, and then another droplet preparation chip is taken to process the next column of the PCR reaction system until all the reaction systems to be tested are processed. Then, the 96-well plate is sealed with an aluminum mold on a heat sealer.

[0155] The sealed 96-well plate is transferred to a qualitative PCR, and PCR reaction is carried out according to the PCR reaction conditions shown in Table 1 above.

[0156] After the PCR is completed, transfer the 96-well plate to a droplet reader, set the droplet reading parameters according to the instruction manual, and pay attention to the fluorescence channel selection: select the FAM channel to detect the nucleic acid of herpes simplex virus type 2, select the VIC channel to detect the internal standard, and start the droplet reading after the setting is completed.

[0157] After the droplet reading is completed, the results are automatically saved. Click "Analyze" to enter the result analysis interface. First, click "Event" to view the total number of droplets in each reaction well. If the number of droplets in a reaction well < 10,000, 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 so, this test is valid.

[0158] If the test is valid, click "1D Amplitude" to view the fluorescence scatter plot of Ch1 (FAM channel). After drawing the threshold line, click "Concentration" to view the nucleic acid concentration of Ch1, in units of copies / μL. Combining with the sample dilution factor, the nucleic acid concentration of the original sample can be calculated. As Figures 6 - 10 shown, among them, Figure 6 、 Figure 7 、 Figure 8 、 Figure 9 and Figure 10 respectively correspond to the sensitivity detection results (FAM channel) of Sample1, Sample2, Sample3, Sample4, and Sample5.

[0159] The sensitivity detection results of Sample1 - Sample5 are shown in Table 3 below:

[0160] Table 3 Sensitivity detection results of Sample1 - Sample5

[0161]

[0162]

[0163] When the sample concentration is greater than 3000 copies / mL, the difference between the sensitivity detection result of this kit and the true concentration ≤ 10%.

[0164] The sample detection range and sensitivity detection experiment of the kit for quantitatively detecting the nucleic acid of herpes simplex virus type 2 provided in this example determine that the minimum detection limit of the kit of this application is 3000 copies / mL, and when it is greater than 3000 copies / mL, the difference between its quantitative test result and the true concentration is also low, verifying the high sensitivity of the kit of this application for detecting herpes simplex virus type 2.

[0165] Example 3

[0166] Example 3 provides an accuracy test for a kit for quantitatively detecting Herpes simplex virus type 2 nucleic acid.

[0167] This kit detects the national reference products P1 - P3 and N1 - N7 of nucleic acid detection reagents for Herpes simplex virus type 1 + 2. Using nucleic acid extraction or purification reagents, nucleic acid extraction and purification are performed on P1 - P3, N1 - N5, and N7, and N6 is directly detected as the nucleic acid extract. Take 5 μL of the extract and add it to the octuplet tube of the PCR reaction system prepared in step S3 to make the total volume 22 μL. Tighten the lid of the octuplet tube, vigorously mix it with a shaker for 15 seconds, and transfer it to the droplet preparation area after centrifuging instantaneously for 15 seconds.

[0168] Mix the PCR reaction system with the added sample using a vortex oscillator. Take out a droplet preparation chip, transfer one column (8) of the PCR reaction system to the sample wells of the chip, add 70 μL of droplet preparation oil to the droplet preparation oil wells, 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.

[0169] Transfer the sealed 96 - well plate to a qualitative PCR instrument and perform a PCR reaction according to the PCR reaction conditions shown in Table 1 above. After the PCR is completed, transfer the 96 - well plate to a droplet reader, set the droplet reading parameters according to the instructions, and note the fluorescence channel selection: select the FAM channel to detect Herpes simplex virus type 2 nucleic acid, select the VIC channel to detect the internal standard, and start droplet reading after setting is completed.

[0170] After the droplet reading is completed, the results are automatically saved. Click "Analyze" to enter the result analysis interface. First, click "Event" to view 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.

[0171] If the test is valid, click "1D Amplitude" to view the fluorescence scatter plot of Ch1. After drawing the threshold line, click "Concentration" to view the nucleic acid concentration of Ch1, with the unit of copies / μL. Combining the sample dilution factor, the nucleic acid concentration of the original sample can be calculated. As Figures 11 - 20 shown, among which, Figure 11 、 Figure 12 、 Figure 13 、 Figure 14 、 Figure 15 、 Figure 16 、Figure 17 , Figure 18 , Figure 19 and Figure 20 respectively correspond to the sample detection results (FAM channel) of P1, P2, P3, N1, N2, N3, N4, N5, N6, and N7.

[0172] The accuracy detection results are shown in Table 4 below:

[0173] Table 4 Accuracy Detection Results

[0174]

[0175] According to the above accuracy detection results, the positive and negative coincidence rates of the accuracy detection of each reference product are 100%, indicating that the accuracy detection of the kit of the present invention meets the requirements.

[0176] The accuracy detection of the kit for quantitatively detecting herpes simplex virus type 2 nucleic acid provided in this example shows that the coincidence rate of the negative reference product is 100% and the coincidence rate of the positive reference product is 100%. It is determined that the accuracy of the kit of this application meets the requirements, the detection accuracy is high, and the quantitative results are accurate.

[0177] Example 4

[0178] Example 4 is a clinical application experiment.

[0179] Eighteen clinical positive samples and eighteen negative samples of herpes simplex virus type 2 were respectively selected. After nucleic acid extraction, the samples were labeled and ensured that the label information was correct, and stored at -80°C. During the experiment, 5 μL of each sample was taken and added to a reaction tube containing the PCR reaction system prepared according to step S3, so that the total volume was 22 μL. It was vigorously shaken and mixed for 15 seconds with an oscillator, and after instantaneous centrifugation for 15 seconds, it was transferred to the droplet preparation area.

[0180] The PCR reaction system with the added sample was mixed using a vortex oscillator. A droplet preparation chip was taken out, and a column (8) of the PCR reaction system was transferred to the sample wells of the chip. 70 μL of droplet preparation oil was added to the droplet preparation oil wells, and it was sent into the droplet preparation instrument for droplet preparation. After the instrument reported that the droplet preparation was completed, the prepared droplets were carefully transferred to a dedicated 96-well plate using a pipette. The droplet preparation chip was discarded, and then another droplet preparation chip was taken to process the next column of the reaction system until all the reaction systems to be tested were processed. Then, the 96-well plate was sealed with an aluminum mold on a heat sealer.

[0181] The sealed 96-well plate was transferred to a qualitative PCR, and the PCR reaction was carried out according to the PCR reaction conditions shown in Table 1 above.

[0182] After the PCR is completed, transfer the 96-well plate to a droplet reader, set the droplet reading parameters according to the instructions, and pay attention to the fluorescence channel selection: select the FAM channel to detect herpes simplex virus type 2 nucleic acid, select the VIC channel to detect the internal standard, and start droplet reading after setting is completed.

[0183] After the droplet reading is completed, the results are automatically saved. Click "Analyze" to enter the result analysis interface. First, click "Event" to view the total number of droplets in each reaction well. If the number of droplets in a reaction well < 10,000, 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 included, this test is valid.

[0184] If the test is valid, click "1D Amplitude" to view the fluorescence scatter plot of Ch1. After drawing the threshold line, click "Concentration" to view the nucleic acid concentration of Ch1, with the unit copies / μL. Combining the sample dilution factor, the nucleic acid concentration of the original sample can be calculated. At the same time, use a third-party herpes simplex virus type 2 detection kit to detect the positive and negative of the tested clinical sample. As Figures 21 - 27 shown, among them, Figure 21 Detection results of positive clinical samples corresponding to numbers 1-8 (FAM channel), Figure 22 Detection results of positive clinical sample corresponding to number 9 (FAM channel), Figure 23 Detection results of positive clinical samples corresponding to numbers 10-18 (FAM channel), Figure 24 Detection results of clinical samples corresponding to numbers 19-27 (FAM channel), Figure 25 Detection results of negative clinical samples corresponding to numbers 19-27 (VIC channel), Figure 26 Detection results of clinical samples corresponding to numbers 28-36 (FAM channel), Figure 27 Detection results of negative clinical samples corresponding to numbers 28-36 (VIC channel).

[0185] The results of the clinical application experiment are shown in Table 5 below:

[0186] Table 5 Results of Clinical Application Experiment

[0187]

[0188]

[0189]

[0190] According to the above experimental results, among the 36 samples, there are 18 positive samples and 18 negative samples of herpes simplex virus type 2, and the consistency of the detected results with the results of the third-party kit is 100%.

[0191] In the clinical application experiment of the kit for quantitatively detecting herpes simplex virus type 2 nucleic acid provided by this embodiment, the final results show that the detection result of the strong positive reference product is positive for the corresponding pathogen, the detection result of the negative precision is negative, and the consistency between the detection results of positive and negative clinical samples and those of the third-party kit reaches 100%. Through the kit of this application, it is possible to identify whether the tested population carries herpes simplex virus type 2 and conduct quantitative detection on it, improving the sensitivity and accuracy of detecting herpes simplex virus type 2.

[0192] It should be understood that although each step in the flowchart of the accompanying drawings is shown in sequence according to the indication of the arrows, these steps do not necessarily have to be executed in the order indicated by the arrows. Unless there is a clear description in this article, there is no strict order restriction for the execution of these steps, and they can be executed in other orders. Moreover, at least a part of the steps in the flowchart of the accompanying drawings may include multiple sub-steps or multiple stages. These sub-steps or stages do not necessarily have to be completed at the same moment, but can be executed at different moments, and their execution order does not necessarily have to be sequential, but can be executed alternately or in turn with at least a part of other steps or sub-steps or stages of other steps.

[0193] Obviously, the above-described embodiments are only a part of the embodiments of this application, rather than all the embodiments. The accompanying drawings show the preferred embodiments of this application, but do not limit the patent scope of this application. This application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of this application more thorough and comprehensive. Although this 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 directly or indirectly using the content of the specification and drawings of this application in other related technical fields is similarly within the scope of the patent protection of this application.

Claims

1. A primer-probe combination for quantitatively detecting Herpes simplex virus type 2 nucleic acid, characterized in that, Comprising: Primers and probes; The primers include an upstream primer of herpes simplex virus type 2, a downstream primer of herpes simplex virus type 2, an upstream primer of an internal control gene, and a downstream primer of an internal control gene; The nucleotide sequence of the upstream primer of herpes simplex virus type 2 is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer of herpes simplex virus type 2 is shown as SEQ ID NO: 2, the nucleotide sequence of the upstream primer of the internal control gene is shown as SEQ ID NO: 3, and the nucleotide sequence of the downstream primer of the internal control gene is shown as SEQ ID NO: 4; The probes include a herpes simplex virus type 2 detection probe and an internal control gene detection probe; The nucleotide sequence of the herpes simplex virus type 2 detection probe is shown as SEQ ID NO: 5, and the nucleotide sequence of the internal control gene detection probe is shown as SEQ ID NO:

6.

2. The primer-probe combination for quantitatively detecting Herpes simplex virus type 2 nucleic acid according to claim 1, characterized in that, The 5' end of the herpes simplex virus type 2 detection probe is labeled with a FAM fluorescent group, and the 5' end of the internal control gene detection probe is labeled with a VIC fluorescent group; the 3' ends of both the herpes simplex virus type 2 detection probe and the internal control gene detection probe are labeled with an MGB quenching group.

3. A kit for quantitatively detecting Herpes simplex virus type 2 nucleic acid, characterized in that, Comprising: A primer-probe mixture, a positive control product, a negative control product, and a droplet digital PCR premix; The primer-probe mixture includes the primer-probe combination for quantitatively detecting herpes simplex virus type 2 nucleic acid as described in claim 1 or 2.

4. The kit for quantitatively detecting Herpes simplex virus type 2 nucleic acid according to claim 3, characterized in that, The positive control product includes an inactivated culture of herpes simplex virus type 2 and a pseudovirus containing an internal standard fragment.

5. The kit for quantitatively detecting Herpes simplex virus type 2 nucleic acid according to claim 3, characterized in that, The negative control product includes TE and a pseudovirus containing an internal standard fragment.

6. The kit for quantitatively detecting Herpes simplex virus type 2 nucleic acid according to claim 3, characterized in that, The final concentration range of the upstream primer of herpes simplex virus type 2 in the PCR reaction system is 0.50 - 0.75 μmol / L, the final concentration range of the downstream primer of herpes simplex virus type 2 in the PCR reaction system is 0.50 - 0.75 μmol / L, the final concentration range of the herpes simplex virus type 2 detection probe in the PCR reaction system is 0.20 - 0.30 μmol / L, the final concentration range of the upstream primer of the internal control gene in the PCR reaction system is 0.41 - 0.60 μmol / L, the final concentration range of the downstream primer of the internal control gene in the PCR reaction system is 0.41 - 0.60 μmol / L, and the final concentration range of the internal control gene detection probe in the PCR reaction system is 0.20 - 0.30 μmol / L.

7. A method for quantitatively detecting Herpes simplex virus type 2 nucleic acid, which uses the kit according to any one of claims 3 to 6 for detection, characterized in that, Comprising the following steps: S1. Collect genital and urinary tract secretions and herpes fluid swab samples; S2. Extract the genital and urinary tract secretions and herpes fluid swab samples with a genital and urinary tract secretion and herpes fluid sample extraction kit to obtain the nucleic acid of the sample to be tested; S3. Prepare the PCR reaction system and dispense the PCR reaction system into corresponding PCR reaction tubes; S4. Add the nucleic acid of the sample to be tested, the negative control product, and the positive control product into the PCR reaction tubes respectively, tighten the tube caps, and perform instantaneous centrifugation; S5. Prepare droplets according to the instruction manual of the droplet digital PCR platform of Bio-rad, transfer the prepared droplets to a 96-well plate dedicated to the droplet digital PCR of Bio-rad, and heat-seal the 96-well plate with an aluminum film; S6. Transfer the heat-sealed 96-well plate to a qualitative PCR, and perform PCR amplification according to the preset PCR reaction conditions to obtain an amplification result; S7. Perform droplet reading and result validity analysis on the amplification result to obtain the copy number of herpes simplex virus type 2 nucleic acid.

8. The method for quantitatively detecting Herpes simplex virus type 2 nucleic acid according to claim 7, characterized in that, The PCR reaction system is prepared through the following steps: Take out the primer-probe mixture and the droplet digital PCR premix, melt them at room temperature and mix them evenly by vortex oscillation, and centrifuge to obtain the PCR reaction system.

9. The method for quantitatively detecting Herpes simplex virus type 2 nucleic acid according to claim 7, characterized in that, The steps of the result validity analysis include: Use the reaction wells with the number of droplets greater than or equal to 10,000 as valid reaction wells, and determine whether the positive control or the negative control exists in each of the valid reaction wells; If the positive control or the negative control exists in each of the valid reaction wells, determine that the test result is valid, and obtain the fluorescence scatter plot and nucleic acid concentration in the FAM channel of the test result.

10. The method for quantitatively detecting herpes simplex virus type 2 nucleic acid according to any one of claims 8 to 9, characterized in that, The method further includes: The concentration range was 3×10 2 ~3×10 5 copies / mL of herpes simplex virus type 2 inactivated culture was used as the sample to be tested; Nucleic acid extraction, sample loading, droplet preparation, and PCR amplification are performed on the sample to be tested, so that the concentration range of the sample to be tested is 3×10 3 ~3×10 5 copies / mL. When the sensitivity detection result of the kit is less than or equal to 10% different from the true concentration.

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