WO2020125295A1

WO2020125295A1 - Primers, probes, kit and detection method for detecting human immunodeficiency virus nucleic acids

Info

Publication number: WO2020125295A1
Application number: PCT/CN2019/119047
Authority: WO
Inventors: 张惠丹; 戴敬; 赵洪玉; 节淳皓
Original assignee: 苏州绘真生物科技有限公司; 苏州德思普生物科技有限公司
Priority date: 2018-12-18
Filing date: 2019-11-18
Publication date: 2020-06-25
Also published as: CN109457052A

Abstract

Disclosed are primers, probes, a kit and a detection method for detecting human immunodeficiency virus nucleic acids. The primers for detecting human immunodeficiency virus nucleic acids comprise: a first primer pair, which comprises a first primer and a second primer, wherein the sequences of the first primer and the second primer are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2; and a second primer pair, which comprises a third primer and a fourth primer, wherein the sequences of the third primer and the fourth primer are respectively as shown in SEQ ID NO.3 and SEQ ID NO.4. A probe pair comprises a first probe and a second probe, wherein the sequences of the first probe and the second probe are respectively as shown in SEQ ID NO.5 and SEQ ID NO.6. The kit comprises a primer group and the probe pair. The detection method comprises: performing, using the kit, PCR amplification on droplets formed by a PCR reaction solution, and on a detected human immunodeficiency virus nucleic acid sample coated with a droplet generation liquid.

Description

Primer, probe, kit and detection method for detecting human immunodeficiency virus nucleic acid

Technical field

The present application relates to the field of molecular biology technology, and more specifically, to the field of RNA technology, in particular to a highly sensitive primer, probe and corresponding kit for detecting human immunodeficiency virus nucleic acid (HIV), and the use of the reagent Method for detecting human immunodeficiency virus.

Background technique

Human immunodeficiency virus (human immunodeficiency virus, HIV) belongs to the family of retroviruses, genus lentivirus. HIV invades and destroys CD4+ T lymphocytes, resulting in damage to the body's immune cells and functions. Eventually, it is complicated by various serious opportunistic infections and tumors. It is an infectious disease with rapid spread, slow onset, and high mortality. According to the statistics of the World Health Organization, there are about 35 million people living with HIV in the world today, and 1 million people died of AIDS in 2016 alone. HIV can be transmitted through sexual, mother-to-child and blood routes, and has an asymptomatic period of 6-8 years after infection. Although there was no obvious clinical manifestation during this period, the virus replicated in the patient's body and destroyed the patient's immune system, until eventually complicated with multiple infectious diseases and even tumors. Through the detection of HIV-RNA, early diagnosis, understanding of disease progression, providing anti-viral evidence, evaluating treatment effect, and guiding adjustment of treatment plan can be provided.

At present, the common detection methods of HIV include viral protein antibody detection, antigen detection, nucleic acid detection and other methods. Viral antigen and antibody detection methods often use enzyme-linked immunosorbent assay (ELISA), that is, specific antigen or antibody is adsorbed on a solid phase carrier, so that it binds to the corresponding antibody or antigen in the sample to be tested, and then enzyme-labeled antigen or Antibody, add substrate to develop color, and finally calculate the content of the antigen or antibody to be tested according to the color depth. However, the amount of virus that has just entered the body is very low. The virus's antigen content is often lower than the detection limit of the ELISA method, and the body needs a period of time to produce antibodies. During this period, the blood antibody test is negative. The antigen antibody is usually after infection. It can only be detected after 3-6 weeks. In recent years, with the development of molecular biology, gene detection technology has been popularized in various laboratories. Especially fluorescent quantitative PCR technology not only has good specificity, but also can increase the sensitivity of detection and shorten virus detection by amplifying viral nucleic acid. Window period.

At present, there are a variety of virus detection kits based on fluorescent quantitative PCR technology at home and abroad, but the domestic detection kit has a high quantitative detection limit and poor sensitivity. The imported high-sensitivity diagnostic kits are too expensive, which increases the cost of medical care.

Therefore, there is an urgent need to develop a kit and a detection method for detecting human immunodeficiency virus nucleic acid with a low detection limit and a high sensitivity to meet the requirements of clinical medicine and detection and diagnosis.

Summary of the invention

The main purpose of the present application is to provide primers and probes for detecting human immunodeficiency virus nucleic acids in order to overcome the deficiencies in the prior art.

Another main objective of the present application is to provide a kit for detecting human immunodeficiency virus nucleic acid.

Another object of the present application is also to provide the use of the aforementioned kit in detecting human immunodeficiency virus nucleic acid.

Another object of the present application is to provide a new method for detecting nucleic acid of human immunodeficiency virus.

In order to achieve the aforementioned object of the invention, the technical solutions adopted in this application include:

The embodiment of the present application provides a primer set for detecting human immunodeficiency virus nucleic acid, which includes:

The first primer pair, which includes the first primer and the second primer, whose sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively;

The second primer pair includes a third primer and a fourth primer, and their sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

The embodiments of the present application further provide a probe pair for detecting nucleic acid of human immunodeficiency virus, which includes:

The first probe, its sequence is shown in SEQ ID NO.5;

The sequence of the second probe is shown in SEQ ID NO.6.

The embodiment of the present application further provides a kit for detecting human immunodeficiency virus nucleic acid, which includes at least one primer set and at least one probe pair, wherein one primer set includes a first primer pair and a second primer pair, The first primer pair includes a first primer and a second primer, the sequences of which are shown in SEQ ID NO. 1, SEQ ID NO. 2, respectively, and the second primer pair includes a third primer and a fourth primer, the sequence of which As shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively, one of the probe pairs is the aforementioned probe pair.

The embodiments of the present application also provide the use of the aforementioned kit in preparing products for detecting human immunodeficiency virus nucleic acids.

Further, the method for detecting human immunodeficiency virus using the product includes:

Provide nucleic acid samples of human immunodeficiency virus to be tested;

A sample processing device is used to mix the nucleic acid sample to be detected with the PCR reaction solution, and the formed mixed solution is wrapped by the droplet generation solution to form droplets containing a single nucleic acid or not;

PCR amplification of the droplet using the primer set and probe pair included in the kit;

Detection of PCR amplification products.

The embodiments of the present application also provide a product containing the aforementioned kit, which is applied to a method for detecting human immunodeficiency virus nucleic acid. The detection method includes:

Provide nucleic acid samples of human immunodeficiency virus to be tested;

Detection of PCR amplification products.

Compared with the prior art, the advantages of this application include:

1) The highly sensitive method for detecting human immunodeficiency virus nucleic acid provided by this application has higher sensitivity and specificity of the detection result, and can effectively reduce the detection cost;

2) This application uses centrifugal force to make the nucleic acid solution and the PCR reaction solution wrapped in the droplet generation solution in the flow channel to form a single-molecule PCR reaction system, which increases the sensitivity of detection and lowers the lower limit of detection, which is conducive to early detection of viral infections;

3) In the present application, because the treated droplets contain only single-molecule nucleic acid templates, the interference received during amplification is reduced, which can avoid generating uncertain results and improve the specificity of the reaction;

4) Unlike other kits that can only detect some common human immunodeficiency virus subtype nucleic acids, the kit provided by this application optimizes the primer design to ensure that the detection of human immunodeficiency virus can cover all known subtypes, and at the same time, In this application, by optimizing primer design, the fragment length of the amplified product is between 100-150bp, and the annealing temperature is between 56-62℃, and the interaction between the designed primers is analyzed by Thermo Fisher primer online tools Perform analysis to ensure that the interaction between primers does not affect the system;

5) This application optimizes the primer probes so that the primer probes for each virus can only detect the virus, and will not interact with the human genome or human hepatitis B virus (HBV) nucleic acid, human herpes virus (EBV) nucleic acid, Common viral nucleic acids such as cytomegalovirus (CMV) nucleic acids cross-react, ensuring the accuracy of positive results.

BRIEF DESCRIPTION

FIG. 1 is a schematic structural diagram of a sample processing device used in a typical embodiment of the present application.

2 is a result of droplet fluorescence detection of the negative control in Example 1 of the present application.

3 is a drop fluorescence detection result of No. 1 positive sample in Example 1 of the present application.

4 is a result of fluorescence detection of the positive sample drop No. 2 in Example 1 of the present application.

FIG. 5 is a result of detecting the fluorescence intensity of the droplet A of Reference A in Example 2 of the present application.

6 is a result of detecting the fluorescence intensity of the droplet B of Reference B in Example 2 of the present application.

7 is a result of detecting the fluorescence intensity of the droplet C of Reference C in Example 2 of the present application.

FIG. 8 is the result of detecting the fluorescence intensity of the droplet of Reference D in Example 2 of the present application.

FIG. 9 is a result of droplet fluorescence intensity detection of HBV, CMV, EBV and HIV positive samples in Example 3 of the present application.

detailed description

The technical solutions of the present application will be explained in more detail below. However, it should be understood that within the scope of the present application, the above technical features of the present application and the technical features specifically described in the following (eg, embodiments) can be combined with each other, thereby forming a new or preferred technical solution. Due to space limitations, I will not repeat them here.

This application will be further described based on specific examples, but it is for illustrative purposes only and does not play a limiting role.

Before describing the example, it is necessary to provide some remarks:

The use of different manufacturers and different batches of reagents will cause differences in experimental results, which is normal.

When conducting small-scale experiments, in order to ensure the repeatability between parallel experiments, it is recommended that after the reagents are configured, they are thoroughly mixed and divided to ensure the uniformity of the reagents in each experiment.

An aspect of an embodiment of the present application provides a primer set for detecting human immunodeficiency virus nucleic acid, which includes:

The first primer pair, which includes a first primer (ie forward primer) and a second primer (ie reverse primer), whose sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively;

The second primer pair includes a third primer (that is, a forward primer) and a fourth primer (that is, a reverse primer), and their sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

Further, the primer sequences contained in the primer set are as follows:

HIV-F1(正向引物)HIV-F1 (forward primer)	5'-GCAGAATTCCAGCCGGAGTC-3'5'-GCAGAATTCCAGCCGGAGTC-3'
HIV-R1(反向引物)HIV-R1 (reverse primer)	5'-CTCCGCATTCACACTTTCTGGT-3'5'-CTCCGCATTCACACTTTCTGGT-3'
HIV-F2(正向引物)HIV-F2 (forward primer)	5'-TAACGTGATCCCCATTGCTG-3'5'-TAACGTGATCCCCATTGCTG-3'

HIV-R2 (reverse primer)

5'-AGATTGTGCCATTGCATTCC-3'

Unlike other kits that can only detect nucleic acids of some common human immunodeficiency virus subtypes, the kit provided by this application optimizes primer design to ensure that the detection of human immunodeficiency virus can cover all known subtypes. At the same time, this application By optimizing the primer design, the fragment length of the amplified product is between 100-150bp, and the annealing temperature is between 56-62℃, and the interaction between the designed primers is analyzed by the Thermo Fisher Primer Analysis Online Tool To ensure that the interaction between primers will not affect the system;

Another aspect of the embodiments of the present application further provides a probe pair for detecting human immunodeficiency virus nucleic acid, which includes:

The first probe, its sequence is shown in SEQ ID NO.5;

The sequence of the second probe is shown in SEQ ID NO.6.

Further, the sequence of the probe pair is as follows:

HIV-Probe1HIV-Probe1	5'-Cy5-AAAAGAAAAGGGGGGATTGGGGGGT-BHQ-3'5'-Cy5-AAAAGAAAAGGGGGGATTGGGGGGT-BHQ-3'
HIV-Probe2HIV-Probe2	5’-ROX-TCCCATCTCTCCTAGTCGC-BHQ-3'5’-ROX-TCCCATCTCTCCTAGTCGC-BHQ-3'

This application optimizes the primer probe so that the primer probe for each virus can only detect the virus, and will not interact with the human genome or human hepatitis B virus (HBV) nucleic acid, human herpes virus (EBV) nucleic acid, megacell Common viral nucleic acids such as viral (CMV) nucleic acids cross-react, ensuring the accuracy of positive results.

Another aspect of the embodiments of the present application also provides a kit for detecting human immunodeficiency virus nucleic acid, which includes at least one primer set and at least one probe pair, wherein one primer set includes a first primer pair and a Two primer pairs, the first primer pair includes a first primer and a second primer, the sequences of which are shown in SEQ ID NO.1, SEQ ID NO.2, respectively, and the second primer pair includes a third primer and a fourth The sequences of the primers are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively, and one of the probe pairs is the aforementioned probe pair.

In some embodiments, the kit further includes a viral internal standard primer pair and an internal standard probe, wherein the viral internal standard primer pair includes a first internal standard primer (ie, internal standard forward primer) and a first Two internal standard primers (that is, internal standard reverse primers), the sequence of the first internal standard primer is shown in SEQ ID NO.7, and the sequence of the second internal standard primer is shown in SEQ ID NO.8 The sequence of the internal standard probe is shown in SEQ ID NO. 9.

Further, the sequences of the internal standard forward primer and the reverse primer contained in the internal standard primer mixed solution in the kit of the present application are as follows:

内标-F(内标正向引物)Internal standard-F (internal standard forward primer)	5'-ATTAACCTTATGTGTGACAT-3'5'-ATTAACCTTATGTGTGACAT-3'
内标-R(内标反向引物)Internal standard-R (internal standard reverse primer)	5'-CATATTCGTCCACAAAATGATTCTG-3'5'-CATATTCGTCCACAAAATGATTCTG-3'

Further, the sequence of the internal standard probe in the internal standard probe solution is: 5'-Cy5-5-CTGTATCGTCAAGGCACT-BHQ-3'.

In some embodiments, the kit also includes conventional components for PCR amplification detection.

Further, the conventional components for PCR amplification detection include a buffer solution for PCR reaction, a mixture of base deoxynucleotide triphosphates (2.5 mM dNTPs), DNA polymerase, RNA reverse transcriptase, and uracil DNA glycosylase , Water and liquid droplets.

Furthermore, the buffer for PCR reaction contains 20 mM potassium chloride, 50 mM PH8.0 Tris-HCl, and 1.5 mM magnesium ions.

Furthermore, dNTPs include dNTP and dUTP.

In some more specific embodiments, the kit specifically includes: PCR reaction buffer, 2.5 mM dNTPs, target nucleotide primer mixed solution, target nucleotide probe solution, internal standard primer mixed solution, internal Standard probe solution, 5U/μL heat-resistant DNA polymerase, 5U/μL RNA reverse transcriptase, 5U/μL uracil DNA glycosylase, water, and droplet generation solution.

Wherein, the target nucleotide primer mixed solution includes the aforementioned first primer pair and second primer pair.

Wherein, the target nucleotide probe solution contains HIV-specific probe pairs.

Wherein, the internal standard primer mixed solution includes an internal standard forward primer and an internal standard reverse primer.

Another aspect of the embodiments of the present application also provides the use of the aforementioned kit in preparing products for detecting human immunodeficiency virus nucleic acids.

Further, the method for detecting human immunodeficiency virus nucleic acid by using the product includes:

Provide nucleic acid samples of human immunodeficiency virus to be tested;

Detection of PCR amplification products.

In some embodiments, the method specifically includes:

Using the primer set and the probe pair included in the kit to perform droplet PCR amplification on the droplet;

The fluorescence intensity of the PCR-amplified droplet is detected, and whether the nucleic acid sample contains human immunodeficiency virus nucleic acid is determined based on the fluorescence intensity.

Further, the sample processing device includes a positioning hole, a sample injection hole, a reagent hole, a reaction hole, and a connecting flow channel. The processing device is made of PC material, with a length of 40 mm, a width of 20 mm, and a thickness of 2 mm. The volume of the injection well is 50 μL, the volume of the reagent well is 50 μL, and the volume of the reaction well is 100 μL.

In some more specific embodiments, the detection method specifically includes: adding a droplet generation solution to the reagent well, adding the nucleic acid sample to be detected and the PCR reaction solution to the injection well, under the action of centrifugal force The liquid in the injection well and the reagent well are mixed through the connecting flow channel, and the formed mixed liquid is wrapped by the droplet generation liquid to form droplets containing a single nucleic acid or not, and enters the reaction well.

Further, the detection method may include: installing the sample processing device on the centrifuge through the positioning hole, adding the droplet generation solution to the reagent hole, and mixing the extracted nucleic acid solution and the PCR reaction solution in proportion to the injection hole. Centrifuge at 3000 rpm clockwise, under the effect of centrifugal force, the liquid in the sample well and the reagent well are mixed through the flow channel, so that the nucleic acid sample is wrapped into the PCR reaction solution to form droplets, and finally collected into the reaction well.

Further, the droplet generation liquid includes oil phase substances such as mineral oil.

In some more specific embodiments, the method may specifically include the following steps:

The PCR reaction solution was removed from the refrigerator in advance and thawed, and after thawing, the mixture was vortexed and centrifuged. Take 10-19 μL of the reaction solution in a PCR tube, add 1-10 μL of the newly extracted nucleic acid sample to it, and mix with a pipette tip to ensure that the total amount of liquid is 20 μL. Pipette 20 μL of the mixed liquid into the injection well, and 20 μL of the droplet generation liquid into the reagent well. Fix the processing device on the centrifuge adapter through the positioning hole, and pay attention to keep the center symmetrical during installation. Centrifuge at 3000 rpm for 3 minutes, so that the nucleic acid sample and the PCR reaction solution flow into the flow channel under the action of centrifugal force, and are surrounded by the droplet generation liquid in the flow channel, forming droplets containing only a single nucleic acid or not.

Transfer the droplets generated in the reaction well to a qPCR tube, put it into a polymerase chain reaction amplifier (PCR amplifier), and perform amplification according to the conditions in Table 1:

Table 1: PCR amplification conditions

The amplified products were analyzed using an 8-channel droplet reading system. First, the background fluorescence intensity was determined according to the blank control without the template added. Droplets exceeding the background fluorescence intensity were set as positive droplets, and the fluorescence intensity of each reaction droplet was measured. And count the number of positive droplets.

By counting the number of positive droplets and determining the volume of the nucleic acid sample added, the amount of human immunodeficiency virus nucleic acid in the nucleic acid sample can be calculated.

This application uses centrifugal force to make the nucleic acid solution and the PCR reaction solution wrapped in the droplet generation solution in the flow channel to form a single-molecule PCR reaction system, which increases the detection sensitivity and lowers the lower detection limit, which is conducive to the early detection of virus infection; In the application, because the treated droplets contain only single-molecule nucleic acid templates, the interference received during amplification is reduced, which can avoid the generation of uncertain results and improve the specificity of the reaction.

Another aspect of the embodiments of the present application also provides a product containing the aforementioned kit, which is applied to a method for detecting human immunodeficiency virus nucleic acid. The method includes:

Provide nucleic acid samples of human immunodeficiency virus to be tested;

Detection of PCR amplification products.

Further, the product includes: performing droplet PCR amplification of the droplet using the primer set and the probe pair included in the kit;

The technical solution of the present application will be further explained below with reference to the drawings and several preferred embodiments, but the experimental conditions and setting parameters therein should not be considered as limitations on the basic technical solution of the present application. And the protection scope of the present application is not limited to the following embodiments.

Example 1

1. The samples used in this example include 2 HIV positive nucleic acid solutions and 1 negative nucleic acid solution.

2. Preparation of PCR reaction system

(1) Remove the PCR reaction solution from the refrigerator in advance and thaw it, after completely thawing, vortex to mix and centrifuge instantly;

(2) Take 19 μL of PCR reaction solution in a PCR tube, add 1 μL of nucleic acid sample to it, and mix with a pipette tip;

(3) Pipette 20μL of mixed liquid into the injection well, and pipette 20μL of droplet generation liquid into the reagent well;

(4) Fix the sample processing device on the centrifuge adapter through the positioning hole, pay attention to keep the center symmetry when installing the processing device;

(5) Centrifuge at 3000 rpm for 3 minutes, so that the nucleic acid sample and the PCR reaction solution flow into the flow channel under the action of centrifugal force, and are surrounded by the droplet generation liquid in the flow channel to form droplets containing only a single nucleic acid or no nucleic acid;

(6) Transfer the droplets generated in the reaction well to the PCR tube.

3. PCR amplification

The amplification is performed in a polymerase chain reaction amplifier (PCR amplifier), and the reaction procedure is shown in the following table:

4. Results analysis

The amplified products were analyzed using an 8-channel droplet reading system (Suzhou Haotong Biotechnology Co., Ltd.). First, the fluorescence intensity of each droplet in the negative control was analyzed to determine the background fluorescence intensity. Then, by setting the droplets that exceed the background fluorescence intensity as positive droplets, the fluorescence intensity of each reaction droplet is measured, and the number of positive droplets is counted. The detection result of each sample takes the order of the detection of each droplet as the abscissa and the fluorescence intensity of the droplet as the ordinate, and plots a scatter plot of the fluorescence intensity of each droplet. The fluorescence intensity threshold line is determined by the background fluorescence value determined by the negative control, and the droplet whose fluorescence intensity exceeds the threshold line is the positive droplet.

Since 1 μL of nucleic acid samples are added in this experiment, the number of positive droplets detected in each sample represents the copy number of HIV virus nucleic acid contained in each 1 μL of nucleic acid sample.

As shown in Figure 2 is the negative control droplet fluorescence intensity detection results.

As shown in Fig. 3, the result of detection of the fluorescence intensity of droplet No. 1 positive sample can be obtained that the nucleic acid load of HIV virus in the nucleic acid sample is 563 copies/μL.

As shown in Fig. 4 is the detection result of the fluorescence intensity of droplet No. 2 positive sample, it can be concluded that the HIV nucleic acid load in the nucleic acid sample is 244 copies/μL.

Example 2

1. The samples used in this example include four concentrations of plasmid quantitative reference containing HIV-specific sequences, of which the plasmid concentrations are A (10copies/μL), B (5copies/μL), C (2copies/μL) and D(1copies/μL).

2. Preparation of PCR reaction system

(2) Take 10 μL of PCR reaction solution in a PCR tube, add 10 μL of nucleic acid sample to it, and mix with a pipette tip;

(6) Transfer the droplets generated in the reaction well to the PCR tube.

3. PCR amplification

4. Results analysis

The amplified products were analyzed using an 8-channel droplet reading system (Suzhou Haotong Biotechnology Co., Ltd.). First, the fluorescence intensity of each droplet in the negative control was analyzed to determine the background fluorescence intensity. Then, by setting the droplets that exceed the background fluorescence intensity as positive droplets, measure the fluorescence intensity of each reaction droplet, and count the number of positive droplets. The detection result of each sample takes the order of the detection of each droplet as the abscissa and the fluorescence intensity of the droplet as the ordinate, and plots a scatter plot of the fluorescence intensity of each droplet. The fluorescence intensity threshold line is determined by the background fluorescence value determined by the negative control, and the droplet whose fluorescence intensity exceeds the threshold line is the positive droplet.

Since 10 μL of nucleic acid samples are added in this experiment, the number of positive droplets detected in each sample represents the number of HIV virus nucleic acid copies contained in each 10 μL of nucleic acid sample.

As shown in Figure 5 is the result of the detection of the fluorescence intensity of the droplet of reference A, it can be concluded that the HIV nucleic acid load in the reference is 10 copies/μL.

As shown in Figure 6 is the result of the detection of the fluorescence intensity of the droplet of reference B, it can be concluded that the HIV viral nucleic acid load in this reference is 5 copies/μL.

As shown in Figure 7 is the result of the detection of the fluorescence intensity of the droplet of reference C, it can be concluded that the HIV nucleic acid load in the reference is 2copies/μL.

As shown in Figure 8 is the result of the detection of the fluorescence intensity of the droplet of the reference D, it can be concluded that the HIV nucleic acid load in the reference is 1 copies/μL.

Example 3

1. The samples used in this example include one sample of human hepatitis B virus (HBV) nucleic acid positive, human herpes virus (EBV) nucleic acid positive, cytomegalovirus (CMV) nucleic acid positive and HIV nucleic acid positive samples.

2. Preparation of PCR reaction system

(6) Transfer the droplets generated in the reaction well to the PCR tube.

3. PCR amplification

4. Results analysis

As shown in Figure 9 from left to right, HBV, CMV, EBV and HIV positive sample droplet fluorescence intensity detection results, it can be concluded that there is no cross-reaction with HBV, CMV, EBV viral nucleic acids, viral nucleic acid load in HIV positive samples 29.3copies/μL.

In summary, with the above technical solutions, the kit of the present application has a low detection limit, high sensitivity and good specificity, the detection method of the present application is simple and fast, the sensitivity and specificity of the test results are higher, and the detection can be effectively reduced cost.

It should be understood that the above embodiments are only to illustrate the technical concept and characteristics of the present application, and the purpose thereof is to enable those familiar with the technology to understand the content of the present application and implement it accordingly, but not to limit the scope of protection of the present application. All equivalent changes or modifications based on the spirit of this application should be covered within the scope of protection of this application.

Claims

A primer set for detecting nucleic acid of human immunodeficiency virus is characterized by comprising:

The first primer pair, which includes the first primer and the second primer, whose sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively;

The second primer pair includes a third primer and a fourth primer, and their sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.
A probe pair for detecting nucleic acid of human immunodeficiency virus is characterized by comprising:

The first probe, its sequence is shown in SEQ ID NO.5;

The sequence of the second probe is shown in SEQ ID NO.6.
A kit for detecting human immunodeficiency virus nucleic acid, characterized by comprising at least one primer set and at least one probe pair, wherein one primer set includes a first primer pair and a second primer pair, the first primer The pair includes a first primer and a second primer, the sequences of which are shown in SEQ ID NO. 1, SEQ ID NO. 2, respectively, and the second primer pair includes a third primer and a fourth primer, the sequence of which is SEQ ID NO. .3. As shown in SEQ ID NO. 4, one of the probe pairs is the probe pair of claim 2.
The kit according to claim 3, further comprising a viral internal standard primer pair and an internal standard probe, wherein the viral internal standard primer pair includes a first internal standard primer and a second internal standard primer The sequence of the first internal standard primer is shown in SEQ ID NO.7, the sequence of the second internal standard primer is shown in SEQ ID NO.8, and the sequence of the internal standard probe is shown in SEQ ID NO .9 shown.
The kit according to claim 3 or 4, further comprising a conventional component for PCR amplification detection; preferably, the conventional component for PCR amplification detection includes a buffer for PCR reaction, a base deoxyribonucleic acid triphosphate Glycoside mixture, DNA polymerase, RNA reverse transcriptase, uracil DNA glycosylase, water and droplet generation liquid; particularly preferably, the PCR reaction buffer contains potassium chloride, Tris-HCl and magnesium ion.
Use of the kit according to any one of claims 3-5 in the preparation of products for detecting human immunodeficiency virus nucleic acids.
The use according to claim 6, wherein the method for detecting human immunodeficiency virus nucleic acid using the product comprises:

Provide nucleic acid samples of human immunodeficiency virus to be tested;

A sample processing device is used to mix the nucleic acid sample to be detected with the PCR reaction solution, and the formed mixed solution is wrapped by the droplet generation solution to form droplets containing a single nucleic acid or not;

PCR amplification of the droplet using the primer set and probe pair included in the kit;

Detection of PCR amplification products.
The detection method according to claim 7, comprising: performing droplet PCR amplification of the droplet using the primer set and the probe pair included in the kit;

Detect the fluorescence intensity of the PCR amplified droplets, and determine whether the nucleic acid sample contains human immunodeficiency virus nucleic acid according to the fluorescence intensity;

And/or, the droplet generation liquid includes an oil phase substance; preferably, the oil phase substance includes a mineral oil;

And/or, the sample processing device includes a sample inlet, a reagent well, a reaction well, and a connecting flow channel; preferably, the detection method includes: adding a droplet generation liquid to the reagent well, and adding the nucleic acid sample to be detected Add the reaction solution with PCR into the injection well, mix the liquid in the injection well and the reagent well through the connecting flow channel under the action of centrifugal force, and make the formed mixed solution be wrapped by the droplet generation liquid to form a single nucleic acid or not The droplets containing nucleic acid enter the reaction well.
A product comprising the kit according to any one of claims 3-5, which is applied to a method for detecting human immunodeficiency virus nucleic acid, the detection method comprising:

Provide nucleic acid samples of human immunodeficiency virus to be tested;

A sample processing device is used to mix the nucleic acid sample to be detected with the PCR reaction solution, and the formed mixed solution is wrapped by the droplet generation solution to form droplets containing a single nucleic acid or not;

PCR amplification of the droplet using the primer set and probe pair included in the kit;

Detection of PCR amplification products.
The product according to claim 9, characterized by comprising: performing droplet PCR amplification of the droplet using the primer set and the probe pair included in the kit;

The fluorescence intensity of the PCR-amplified droplet is detected, and whether the nucleic acid sample contains human immunodeficiency virus nucleic acid is determined based on the fluorescence intensity.