CN111719015A - A detection kit for human immunodeficiency virus HIV-1 - Google Patents

Abstract

The invention discloses a detection kit for human immunodeficiency virus HIV-1, which belongs to the technical field of HIV molecular biological detection. The kit includes upstream primers, downstream primers, probes used with primers, negative control template, positive control template, standard template, RAA isothermal nucleic acid amplification reagent, 10x NEB Buffer2.1, 40U/μL RNase inhibitor , 2M DTT and 2μM EnGen Lba Cpf1 nuclease. The kit is used for detection, the reaction speed is fast, and the whole detection process takes less than 2 hours; and only the virus DNA needs to be extracted, the operation steps are few and simple, which can effectively avoid pollution; at the same time, the detection sensitivity is high, and 10 copies can be detected. Viral DNA greatly improves work efficiency and reduces detection costs.

Description

A detection kit for human immunodeficiency virus HIV-1

technical field

The invention relates to a detection kit for human immunodeficiency virus HIV-1, which belongs to the technical field of HIV molecular biology detection.

Background technique

AIDS (acquired immune deficiency syndrome, AIDS) is a very harmful viral infectious disease, a serious threat to global public health. At present, about 37 million people in the world have been infected with human immunodeficiency virus (human immunodeficiency virus, HIV), and about 1.8 million new infections are added every year. Since the first discovery of HIV/AIDS among drug users in Ruili City, Yunnan Province in 1989, drug use has always been one of the main ways of contracting AIDS in my country, especially among injecting drug users due to high-risk behaviors such as sharing needles and cotton balls. The existence of AIDS makes it more likely to become an affected population of AIDS. The distribution of drug users and their HIV infection in my country is mostly related to geographical distribution. Due to its proximity to the "Golden Triangle", a famous drug production base, drug use is more common in Yunnan than in the mainland.

Highly active antiretroviral therapy (HAART) can effectively control HIV-1 virus replication in plasma and inhibit HIV-1 virus to undetectable levels, but ART cannot clear the latent virus reservoir. The latent virus reservoir is the main source of viral re-emergence after interruption of ART treatment and a major obstacle to the clearance of latent HIV-1 virus. In the SIV (simian immunodeficiency virus) model of AIDS, it was confirmed that the virus reservoir was established within 1-3 days after virus infection, so even HAART treatment after 3 days of HIV-1 virus infection could not prevent the establishment of persistent infection. Therefore, it is necessary to develop a new detection method for HIV virus latent reservoirs to lay a technical foundation for the "functional" cure of HIV/AIDS patients.

Molecular (nucleic acid)-based diagnostic tests have numerous advantages over protein immunoassays, especially in terms of detection sensitivity and specificity. However, traditional nucleic acid-based tests (NATs) require the processing of large numbers of samples, well-trained operators, and specialized equipment. On-site detection of pathogens and other disease markers can improve the quality of care and reduce healthcare costs. The gold standard for HIV diagnosis is DNA PCR, which detects proviral DNA present in peripheral blood mononuclear cells. HIV DNA testing is reliable even when HIV viral load is suppressed to very low levels. Unfortunately, HIV DNA PCR is not suitable for performing in poor areas because PCR requires expensive equipment, electricity, dedicated laboratory space, and trained technicians. Therefore, there is a need for a DNA-based point-of-care HIV testing technology that can provide results quickly, enabling more patients to understand their HIV status and start treatment sooner. Compared with other conventional molecular methods, isothermal amplification technology has the advantages of rapidity, simple operation and low cost, and is very suitable for clinical application.

At present, the diagnosis of viral infection generally includes virus isolation and identification, direct detection of viral nucleic acid and antigen, and detection of specific antibodies, which is not only time-consuming, but also requires high professional standards of medical equipment and operators. Therefore, it is very important to develop an intelligent diagnostic device that does not need to rely on expensive equipment and medical staff, especially for developing countries where viral infections often run rampant. The conventional HIV infection detection method is to detect the presence of antiviral antibodies in human serum or plasma by enzyme-linked immunosorbent assay (ELISA). During the month ("window period"), antibodies may not be detected at all; while the detection of viral nucleic acid can greatly shorten the detection window period.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a primer, a probe and a detection kit for qualitative detection of human immunodeficiency virus HIV DNA. To characterize HIV DNA in samples to be tested. The primers, probes and detection kits provided by the invention can not only be used for clinical detection of HIV, but also can be used for qualitative analysis of HIV DNA in HIV-infected patients, and will be used in the prevention and control of HIV/AIDS in my country. Play an important role.

The above purpose is achieved through the following technical solutions:

A detection kit for human immunodeficiency virus HIV-1, including DNA isothermal amplification-CRISPR technology detection primers and probes:

The specific primers of the HIV in vitro isothermal nucleic acid amplification-CRISPR, including the upstream primer HIV-RAA_F and the downstream primer HIV-RAA_R;

Upstream primer HIV-RAA_F: TACTTCAAGAACTGCTGACATCGAGCTTGCTAC; (SEQ ID NO. 1).

Downstream primer HIV-RAA_R: CGCCACTGCTAGAGATTTTCCACACTGACT; (SEQ ID NO. 2).

The probe nucleotide sequence is: TTTCCACACTGACTAAAAGGGTCT (SEQ ID NO. 3).

The present invention also provides a ssDNA-FQ reporter gene probe used in combination with the above primers, and the nucleotide sequence of the probe is: FAM-TTATT-BHQ1 (SEQ ID NO. 4).

Further, preferably, the detection kit for human immunodeficiency virus HIV-1 of the present invention further includes a negative control template, a positive control template, RAA isothermal nucleic acid amplification reagent, 10x NEB Buffer2.1, and 40U/μL of RNase inhibitor , 2M DTT and 2μM EnGen Lba Cpf1 nuclease.

The negative control template is Ribozyme-free water and pUC57-HBV plasmid; the positive control template is pUC57-HIV LTR plasmid. When the in vitro isothermal nucleic acid amplification-CRISPR detection system reacts with a negative control template, the template is Ribozyme-free water and pUC57-HBV plasmid of known concentration; when the in vitro isothermal nucleic acid amplification-CRISPR detection system reacts with a positive control template When the control template is reacted, the template is the pUC57-HIV LTR plasmid; when the in vitro isothermal nucleic acid amplification-CRISPR detection system is reacted with the sample template to be tested, the template is extracted from the blood of a suspected HIV-infected person. of viral genomic DNA. The negative control template, positive control template and sample template to be tested are subjected to in vitro isothermal nucleic acid amplification-CRISPR detection using the primers and probes provided by the present invention, and after the reaction is terminated, the excitation wavelength is 535 nm and the emission wavelength is 575 nm on a VICTOR Nivo microplate reader. , collect the fluorescence value, subtract the fluorescence value of the blank hole from the template fluorescence value of the sample to be tested to obtain the final fluorescence value, when the final fluorescence value of the sample to be tested is less than or equal to 0, it is interpreted that no virus is detected (the test result is negative).

Further, preferably, the RAA isothermal nucleic acid amplification reagent of the present invention includes a basic reaction solution, a basic reaction unit containing lyophilized powder and a magnesium acetate solution.

Further, preferably, the RAA isothermal nucleic acid amplification system of the kit of the present invention is:

Basal buffer, 25.0 μL;

Upstream primer HIV-RAA_F (10μmol/L), 2.0μL;

Downstream primer HIV-RAA_R (10μmol/L), 2.0μL;

Template DNA, 1.0 μL;

Purified water, 17.5 μL;

Magnesium acetate solution, 2.5 μL;

A total of 50.0 μL.

Further, preferably, the nucleic acid amplification procedure of the kit of the present invention: 37°C for 40 minutes; after the reaction, add 50 μL of phenol/chloroform (1:1) to each reaction tube, use a vortex shaker to fully Shake evenly and centrifuge at 12,000 rpm for 1 minute.

Further, preferably, the CRISPR DNA detection system of the kit of the present invention is:

10x NEB Buffer2.1, 2.0μL;

EnGen Lba Cpf1 Nuclease, 2.0 μL;

LTR-crRNA probe (10μmol/L), 1.0μL;

RAA amplification product, 4.0 μL;

ssDNA-FQ reporter gene probe (100μmol/L), 0.2μL;

RNase inhibitor, 0.25 μL;

DTT, 0.1 μL;

Ribozyme-free water, 10.45 μL;

A total of 20.0 μL.

Further, preferably, the detection procedure of the kit is as follows: reaction at 37° C. for 40 minutes, and heating at 98° C. for 5 minutes to terminate the reaction.

The present invention provides a ssDNA-FQ reporter gene probe used for detecting HIV DNA whose 5' end is labeled with a reporter fluorescent group, and the 3' end is labeled with a quenching fluorescent group. Among them, FAM is a 6-carboxyfluorescein reporter fluorophore, and BHQ1 is a black hole quenching fluorophore.

The specific principle of the present invention is that when the CRISPR-Cas12a (Cpf1) system cuts the targeted double-stranded DNA, the DNase activity of Cpf1 will be activated, and the enzyme can non-specifically cut single-stranded ssDNA in trans; The CRISPR-Cpf1 system targeting the DNA and a non-specific ssDNA fluorescent reporter group are delivered intracellularly. Once the target DNA is detected, the CRISPR-Cpf1 system will be activated, and the fluorescent reporter group will be degraded, releasing a fluorescent signal. DNA or RNA samples are amplified by isothermal amplification methods such as recombinase polymerase amplification of RAA; then, with the help of specific guideRNAs, the CRISPR-associated protein Cpf1 binds to the amplified target sequence, passing through the reporter's bypass nucleic acid cleavage activity to detect the resulting results.

Design specific primers for isothermal nucleic acid amplification and LTR-crRNA probe and ssDNA-FQ reporter gene probe for CRISPR detection against HIV LTR DNA sequence, using FAM group as the reporter fluorescence of ssDNA-FQ reporter gene group, BHQ1 as the quenching fluorophore of the ssDNA-FQ reporter gene; pUC57-HBV plasmid and pUC57-HIV LTR plasmid were synthesized in Kunming Qingke Biotechnology; probe was synthesized in Shanghai Shenggong; RAA was purchased in Jiangsu Qitian Gene Co., Ltd. Isothermal nucleic acid amplification kit; finally constructed detection primers, probes and detection kits suitable for the combination of isothermal nucleic acid amplification and CRISPR detection of HIV DNA.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention designs a highly specific in vitro isothermal nucleic acid amplification primer and a CRISPR detection probe, and on this basis constructs a detection kit capable of detecting HIV DNA, the kit has specificity, sensitivity, rapidity and high efficiency.

(2) The CRISPR detection technology of the present invention can detect the highest fluorescence value at 37° C. for 40 minutes (Fig. 1), which is faster than the detection time using the traditional RT-qPCR method.

(3) In the early stage of human infection with HIV, the virus content in the blood is low, and the in vitro nucleic acid isothermal amplification-CRISPR technology detection kit developed by the present invention has good detection sensitivity and is suitable for the detection of early clinical samples. The sensitivity test results show that the detection limit of the in vitro nucleic acid isothermal amplification-CRISPR technology detection kit developed by the present invention is 10 copies of HIV virus DNA (Fig. 2), while the detection limit of RT-qPCR is generally 50 copies. It shows that the detection sensitivity of the present invention is 5 times higher than that of ordinary RT-qPCR.

(4) The in vitro nucleic acid isothermal amplification-CRISPR technology detection kit developed by the present invention has strong specificity. As shown in FIG. 3 , the primers and probes involved in the present invention can only generate fluorescence values in HIV DNA samples without cross-reacting with HBV DNA.

(5) The in vitro nucleic acid isothermal amplification-CRISPR technology detection kit developed by the present invention has a fast reaction speed, and the whole detection process can be completed in less than 2 hours, and can directly determine the pending detection by detecting the fluorescence value without agarose gel electrophoresis Whether the sample has HIV DNA, it takes less time than the traditional electrophoresis detection method, which greatly improves the work efficiency.

(6) Since the HIV LTR sequence is highly conserved, it is usually used as a target gene for HIV virus-specific detection. The present invention conducts primer design for the HIV LTR sequence, and then utilizes the in vitro nucleic acid isothermal amplification developed by the present invention in the application example- The CRISPR technology detection kit detects the blood samples of HIV/AIDS patients collected by the Second People's Hospital of Dali City, and the detection results show that the detection kit developed by the present invention can effectively detect HIV DNA in the blood samples of HIV/AIDS patients. The compliance rate is 100%.

Description of drawings

Fig. 1 is the time-response curve of the present invention; the X-axis is the reaction time for detecting HIV DNA, and the Y-axis is the fluorescence value.

Figure 2 Sensitivity test of the in vitro nucleic acid isothermal amplification-CRISPR technology kit developed by the present invention; wherein, 1 ng of template HIV DNA is equal to 10 ⁸ aM.

Fig. 3 The specificity test of the in vitro nucleic acid isothermal amplification-CRISPR technology kit developed by the present invention.

Detailed ways

Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. If no specific technology or condition is indicated in the examples, the technology or condition described in the literature in the field or the product specification is used. If the materials or equipment used are not marked with the manufacturer, they are all conventional products that can be obtained through purchase.

(1) Experimental materials

The blood samples of HIV/AIDS patients were collected from the scientific research project "Clinical Trial of Nutritional Treatment of AIDS Patients with Nutritional Supplementation of Whole-Nutrition Formula Foods" carried out in the Second People's Hospital of Dali City. Six compulsory drug rehabilitation centers Longchuan branch. Blood samples were divided into 200 μL/tube and stored in -80°C refrigerator.

(2) Reagents and instruments

10x NEB Buffer2.1 and EnGen Lba Cas12a(Cpf1) nuclease were purchased from NEB Company; Plasmid Mini Kit, Escherichia coli DH5α Competent Cell, Blood Genomic DNA Extraction Kit were purchased from Tiangen Biochemical Technology Co., Ltd.; Phenol/chloroform (1:1) was purchased from Solebao Company; RAA nucleic acid detection kit (Item No.: B00000) was purchased from Jiangsu Qitian Gene Company. The pUC57-HIV LTR plasmid and pUC57-HBV plasmid were synthesized by Kunming Qingke Biotechnology Co., Ltd. and stored in -20°C refrigerator.

384-well plate; VICTOR Nivo microplate reader; dry thermostatic metal bath OSE-96 (Tiangen Biochemical Technology Co., Ltd.); desktop centrifuge 1-14 (Sigma).

(3) Design primers and probes

Specific primers and LTR-crRNA probes for in vitro isothermal nucleic acid amplification were designed for HIV LTR sequences, and ssDNA-FQ reporter gene probes were also designed. The reporter gene probes used FAM and BHQ1 as the reporter fluorophore and quencher, respectively. Fluorophore. Primer and probe sequences are shown in Table 1.

Table 1 Sequence information of primers and probes used in PALV qRT-PCR detection

(4) Synthesis of template plasmid

According to the LTR sequence (806bp) and the synthesized HBV sequence (989bp) of HIV HxB2 strain, pUC57-HIV LTR plasmid and pUC57-HBV plasmid were synthesized in Kunming Qingke Biotechnology Company, respectively.

The synthesized plasmids were transformed into Escherichia coli DH5α competent cells (Tiangen Biochemical Technology Co., Ltd.), and positive clones were screened for plasmid extraction. The template plasmid was extracted according to the instructions of "plasmid extraction kit" (Tiangen Biochemical Technology Co., Ltd.).

(5) Optimize the CRISPR detection system

(6) Through repeated experiments, the reaction system of CRISPR detection was optimized, and the total reaction system used was determined to be 20 μL. The required components, corresponding concentrations, and corresponding amounts are shown in Table 2. Using 10ng final concentration of pUC57-HIV LTR plasmid as template, the LTR crRNA probe concentrations (0.25, 0.5, 0.75 and 1.0 μmol/L) and ssDNA-FQ reporter gene probe concentrations (0.2, 0.4, 0.6, 0.8 and 1.0 μmol/L) for HIV DNA detection in a 20 μL reaction system, the best results correspond to the final concentrations of LTR crRNA probe and ssDNA-FQ reporter gene probe of 0.5 μmol/L and 1 μmol/L, respectively. If the reaction system is adjusted, it should be ensured that the final concentrations of the LTR crRNA probe and the ssDNA-FQ reporter gene probe in the system are 0.5 μmol/L and 1 μmol/L, that is, better detection results can be obtained.

Table 2 CRISPR detection HIV DNA reaction system

Reaction system components Dosage (μL) Final concentration 10×NEB Buffer2.1 2.0 EnGenLba Cas12a (2μmol/L) 2.0 DTT(2mol/L) 0.1 RNase inhibitors (40U/μL) 0.25 LTR-crRNA Probe (10μmol/L) 1.0 0.5μmol/L ssDNA-FQ reporter gene probe (100μmol/L) 0.2 1μmol/L Template (100ng/μL) 0.1 Ribozyme-free water 14.35 total 20.0

(6) Optimizing reaction conditions for isothermal nucleic acid amplification in vitro

Perform in vitro nucleic acid isothermal amplification according to the operating instructions of the RAA in vitro nucleic acid isothermal amplification kit; in order to obtain the maximum amount of target amplification products, the optimal reaction conditions for RAA isothermal nucleic acid amplification are determined as: 37 °C for 40 minutes.

(7) Optimize the volume of RAA amplification products used in the in vitro nucleic acid isothermal amplification-CRISPR technology detection kit reaction system: through repeated experiments, optimize the in vitro nucleic acid isothermal amplification-CRISPR technology detection kit detection reaction system, and determine the reaction used The total system is 20 μL, and the required components and corresponding amounts are shown in Table 3.

Using the RAA amplification product as a template, the use volume (1.0, 2.0, 3.0, 4.0, and 5.0 μL) was optimized. HIV DNA detection was performed in a 20 μL reaction system, and the optimal amount of RAA amplification product was 4.0 μL. Test results.

Table 2 In vitro nucleic acid isothermal amplification-CRISPR technology detection kit reaction system

(8) The time-response curve of the present invention takes 10 ng HIV DNA as a template, respectively detects the fluorescence values when the reaction time is 10, 20, 30, 40, 50 and 60 minutes, and establishes the time-response curve of the present invention. Taking the reaction time for HIV DNA detection as the X-axis and the final fluorescence value as the Y-axis, the time-response curve is shown in Figure 1. It can be seen from the figure that CRISPR-Cpf1, LTRcrRNA-1 and 10ng HIV DNA are used in combination at 37°C After 40 minutes of reaction, the highest fluorescence value can be detected.

(9) Sensitivity analysis

Using the HIV DNA detection primers, probes and detection kits involved in the present invention, according to the optimized reaction system and reaction conditions, the copy numbers are respectively 10 ⁶ aM, 10 ⁵ aM, 10 ⁴ aM, 10 ³ aM, 10 ² After isothermal amplification of aM, 10 ¹ aM and 10 ⁰ aM of template HIV LTR by nucleic acid in vitro, 4 μL of the amplified product was taken for sensitivity analysis. The detection limit of the detection primers, probes and kits involved in the present invention is 10 copies. (see Figure 2). It can be seen from the figure that RAA isothermal nucleic acid amplification technology combined with CRISPR-Cpf can detect 10 copies of HIV virus DNA (for template DNA, 1 ng is equal to 10 ⁸ aM).

(10) Specificity analysis

Using the HIV DNA detection primers, probes and detection kits involved in the present invention, according to the optimized reaction system and reaction conditions, take 4 μL of the isothermally amplified product of nucleic acid in vitro for specificity analysis. The HIV DNA detection primers involved in the present invention , probes and detection kits can specifically detect HIV DNA without cross-reacting with HBV DNA, as shown in Figure 3. It can be seen from the figure that the combination of RAA isothermal nucleic acid amplification technology and CRISPR-Cpf is specific good detection method.

Applications

Detecting HIV/AIDS patients by using the detection kit developed by the present invention

Using the HIV DNA detection primers, probes and detection kits involved in the present invention, according to the optimized reaction system and reaction conditions, 30 blood samples of HIV/AIDS patients and 30 blood samples of drug addicts who were not infected with HIV were collected. detection. 100 μL of blood was taken to extract genomic DNA. During the operation, the negative control template, positive control template and sample template to be tested were simultaneously subjected to in vitro nucleic acid isothermal amplification and CRISPR detection in different reaction wells. The primers, probes and detection kits of the invention can effectively detect all positive samples, and the detection results of drug addicts who are not infected with HIV are all negative, and the coincidence rate is 100%. The HIV DNA detection kit developed by the present invention detects pathogenic nucleic acids. Therefore, when the host is infected but has not yet produced antibodies, the HIV DNA in vitro nucleic acid isothermal amplification primers, CRISPR-related probes and detection kits developed by the present invention have Good sensitivity and reliability.

The primer sequence derived from the primer pair of the present invention also belongs to the protection scope of the present invention; the derived sequence refers to the one to ten base substitutions, deletions or additions obtained on the basis of SEQ ID NO.1 to SEQ ID NO.4. primer sequences.

The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments. The above-mentioned embodiments and descriptions only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have Various changes and modifications fall within the scope of the claimed invention. The claimed scope of the present invention is defined by the appended claims and their equivalents.

sequence listing

<110> The First Affiliated Hospital of Kunming Medical University

<120> A detection kit for human immunodeficiency virus HIV-1

<130> 20200521

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<213> LTR of HIV HxB2 strain (HIV)

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tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60

cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120

tgacctttgg atggtgctac aagctagtac cagttgagcc agataagata gaagaggcca 180

ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240

agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300

agctgcatcc ggagtacttc aagaactgct gacatcgagc ttgctacaag ggactttccg 360

ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420

cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480

gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540

tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600

agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacctgaaag 660

cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720

caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780

aggagagaga tgggtgcgag agcgtc 806

<210> 1

<211> 989

<212> DNA

<213> Synthetic HBV (HIV)

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ggattgggga ccctgcgctg aacatggaga acatcacatc aggattccta ggacccctgc 60

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actcgtggtg gacttctctc aattttctag gggggaccac cgtgtgtctt ggccaaaatt 180

cgcagtcccc aacctccaat cactcaccaa cctcctgtcc tccaacttgt cctggttatc 240

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aagatggggt tactctttaa atttcatggg ctatgtcatt ggatgttatg ggtcattgcc 780

acaagatcac atcagacaga aaatcaaaga atgttttaga aaacttcctg ttaacaggcc 840

tattgattgg aaagtctgtc aacgtattgt gggtcttttg ggttttgctg ccccttttac 900

acaatgtggt tatcctgctt taatgccctt gtatgcctgt attcaatcta agcaggcttt 960

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Claims (8)

1. a detection kit of human immunodeficiency virus HIV-1, is characterized in that: comprise the isothermal amplification-CRISPR technology detection primer and probe of DNA; The specific primers of the HIV in vitro isothermal nucleic acid amplification-CRISPR, including the upstream primer HIV-RAA_F and the downstream primer HIV-RAA_R; Upstream primer HIV-RAA_F: TACTTCAAGAACTGCTGACATCGAGCTTGC TAC; Downstream primer HIV-RAA_R: CGCCACTGCTAGAGATTTTCCACACTGACT; The probe nucleotide sequence is: TTTCCACACTGACTAAAAGGGTCT. 2. the detection kit of human immunodeficiency virus HIV-1 according to claim 1, is characterized in that: The nucleotide sequence of the ssDNA-FQ reporter gene probe used in combination with the primers is: FAM-T TATT-BHQ1. 3. the detection kit of human immunodeficiency virus HIV-1 according to claim 1 or 2, is characterized in that: also comprises negative control template, positive control template, RAA isothermal nucleic acid amplification reagent, 10x NEB Buffer2.1, 40U /μL of RNase inhibitor, 2mol/L of DTT and 2μmol/L of EnGen Lba Cpf1 nuclease; Described negative control template is Ribozyme-free water and pUC57-HBV plasmid; The positive control template is pUC57-HIV LTR plasmid. 4. the detection kit of human immunodeficiency virus HIV-1 according to claim 3, is characterized in that: described RAA isothermal nucleic acid amplification reagent comprises basic reaction solution, the basic reaction unit that freeze-dried powder is housed and magnesium acetate solution. 5. the detection kit of human immunodeficiency virus HIV-1 according to claim 4, is characterized in that: the RAA isothermal nucleic acid amplification system of described test kit is: Basal buffer, 25.0 μL; Upstream primer HIV-RAA_F 10μmol/L, 2.0μL; Downstream primer HIV-RAA_R 10μmol/L, 2.0μL; Template DNA, 1.0 μL; Purified water, 17.5 μL; Magnesium acetate solution, 2.5 μL; A total of 50.0 μL. 6 . The detection kit for human immunodeficiency virus HIV-1 according to claim 3 , wherein the nucleic acid amplification procedure of the kit is: 37° C. for 40 minutes; after the reaction is completed, each reaction tube is added with 50 μL of phenol/chloroform (1:1), fully shaken with a vortex shaker, and centrifuged at 12,000 rpm for 1 minute. 7. the detection kit of human immunodeficiency virus HIV-1 according to claim 3, is characterized in that: the CRISPR DNA detection system of described test kit is: 10x NEB Buffer2.1, 2.0μL; EnGen Lba Cpf1 Nuclease, 2.0 μL; LTR-crRNA probe 10μmol/L, 1.0μL; RAA amplification product, 4.0 μL; ssDNA-FQ reporter gene probe 100μmol/L, 0.2μL; RNase inhibitor, 0.25 μL; DTT, 0.1 μL; Ribozyme-free water, 10.45 μL; A total of 20.0 μL. 8 . The detection kit for human immunodeficiency virus HIV-1 according to claim 1 , wherein the reaction is terminated at 37° C. for 40 minutes and heated at 98° C. for 5 minutes. 9 .

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