Kit for rapidly and quantitatively detecting integrated HIV (Human Immunodeficiency Virus) previrus genome and application thereof

Abstract

The invention discloses a kit for rapidly and quantitatively detecting an integrated HIV (Human Immunodeficiency Virus) previrus genome and application of the kit. The kit comprises an anchor primer in an Alu region in an HIV virus, and degenerate primers in gag, nef and LTR regions of an HIV sequence. According to the kit disclosed by the invention, after pre-amplification, the integrated copy number AU-LTR detected by the QPCR is obviously improved compared with AU-gag and AU-nef, and the capture efficiency is higher. Meanwhile, the kit provided by the invention can be used for carrying out three-generation sequencing on a detected sample with a relatively high integration level on the basis of Alu-LTR capture and detection of an integrated copy number, and can be used for carrying out sequence composition and genome position analysis on a storage library of HIV (Human Immunodeficiency Virus).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a kit for rapid quantitative detection of integrated HIV provirus genome and application thereof. BACKGROUND

[0002] AIDS is a major infectious disease threatening human life safety caused by human immunodeficiency virus type 1 (HIV-1) infection. According to the Joint United Nations Programme on HIV / AIDS (UNAIDS), there were 3770 million HIV / AIDS patients worldwide at the end of 2020, 1.5 million new HIV infections occurred in the year, and 27.5 million people were receiving anti-retroviral therapy (ART; commonly known as “cocktail therapy”).

[0003] The first prerequisite for containing and preventing AIDS is rapid and efficient diagnosis and detection. There are more than 100 methods for detecting HIV at present, which can be generally divided into serological diagnosis and virological diagnosis. Serological detection is the main method for early HIV diagnosis. Commonly used methods for virological diagnosis of HIV infection include co-culture, p24 antigen detection and viral nucleic acid detection. The co-culture method has poor sensitivity, long operation time, complex operation and high cost, and can only be performed in a specific P3 laboratory, so it is not suitable for clinical use. The p24 antigen detection can detect soluble p24 antigen in blood after the virus begins to replicate, but false positives may occur. Moreover, HIV-1 p24 antigen detection negative only indicates no reaction in this test, and cannot rule out HIV infection.

[0004] These HIV diagnosis methods all have their own limitations. In recent years, comprehensive and efficient anti-retroviral therapy HAART can reduce the incidence and mortality of AIDS, and reduce the HIV-1 RNA in the plasma of infected persons to a level that cannot be detected by conventional methods. Only by using a method with higher sensitivity can viral genes and low-level viral replication be detected, which makes HIV RNA detection also show great limitations. However, the fact that HIV RNA cannot be detected does not mean that the virus in the body has been cleared.

[0005] Although antiretroviral therapy can control viremia and improve health outcomes, HIV seropositive individuals who continue to receive antiretroviral therapy are still at high risk of developing immune dysfunction and inflammatory diseases compared with uninfected individuals. Due to the presence of quiescent CD4+ T lymphocyte host genome, viral rebound will occur after treatment interruption, even after years of suppressive antiretroviral therapy. The main reason for viral rebound is that HIV establishes a viral "reservoir" in the body of the infected person. Over the past 30 years, research has focused on studying the HIV "reservoir" to eradicate the "reservoir" (cure) or maintain the HIV seropositive individuals with suppressed viremia (functional cure) as the main research goal. One of the core strategies for eradication (cure) is to use latency reversing agents (LRAs) in combination with vaccines, antibodies and / or cellular immunotherapy to induce HIV viral expression to facilitate immune clearance of virus-infected cells. The main strategies for functional treatment include the use of long-acting broadly neutralizing antibodies (passive immunotherapy), vaccination (active immunotherapy), modification of host cells to prevent viral replication (gene therapy) or permanent silencing of the provirus ("block and lock"). The common point of all these treatment or functional treatment methods is the need to carefully identify and measure the "reservoir".

[0006] The HIV "reservoir" includes active and latent. When the HIV viral load level in the patient's body is below the detection limit, the main latent viral "reservoir" in the body exists in quiescent central memory CD4+ T lymphocytes. At this time, the viral reservoir level in the patient's body can be roughly obtained by HIV-1 DNA quantification. However, the HIV-1 DNA in virus-infected cells exists in various forms, including linear non-integrated, 1-LTR circular, 2-LTR circular and integrated, of which integrated HIV-1 DNA accounts for only a small part of the total DNA (up to 1 / 25), and more importantly, only integrated HIV viral genome has high transcriptional activity and is the main form of viral reservoir, which will rebound rapidly from the reservoir once the drug is stopped. At present, more literature reports only use real-time PCR method to quantify HIV-1 total DNA and determine the content of viral reservoir. This detection cannot accurately quantify the integrated HIV "reservoir" in the genome because it will cover various forms of HIV-1 DNA. Therefore, it is a new topic to be solved to develop a method and kit for accurately quantifying and locating integrated HIV-1 viral genome.

[0007] The most similar scheme to the present technical scheme in the prior art is the Alu-gag PCR method for detecting the copy number of integrated HIV genome. The method steps and process of this technology are as follows:

[0008] (1) Isolation and purification of genomic DNA from infected cells.

[0009] (2) First amplification using primers located on both sides of the Alu sequence and the HIV structural gene gag to amplify the genetic sequence. Only copies of the integrated form of the HIV genome will be exponentially amplified. Uninfected genomic DNA will only be linearly amplified in this step.

[0010] (3) When using fluorescent quantitative PCR, use forward primer R and reverse primer U5, which are located on both sides of the viral LTR region, and use a plasmid with a known copy number as a standard template, combined with a probe for real-time PCR to quantify the number of HIV integrated copies.

[0011] There are also studies on the analysis method of the integration site of lentiviral vector in CAR-T cells, which is to use specific primers of the LTR region of lentiviral vector for single-direction PCR to capture the LTR region inserted into the genome, and then use two sets of adapter primers for nested PCR amplification to obtain the integration site sequence. The specific embodiment is:

[0012] According to the specific 5' end of the 5' LTR region of the lentiviral vector, design an amplification primer labeled with biotin; (1) using CAR-T genomic DNA as a template, linear single-direction PCR amplification, using streptavidin magnetic beads to collect the amplification product;

[0013] (2) Design a single-stranded DNA linker (ssLC) with 5' phosphorylation and 3' dideoxy, and use RNA ligase to connect the 5' end of the single-stranded DNA linker to the 3' end of the amplification product of step (1);

[0014] (3) First round of PCR amplification: according to the sequence of the DNA linker and the 5' end LTR region of the lentiviral vector, design the first pair of primers to perform PCR amplification with the connection product of step (2) as the template;

[0015] (4) Second round of PCR amplification: according to the sequence of the DNA linker and the 5' end LTR region of the lentiviral vector, design the second pair of primers to perform PCR amplification with the amplification product of step (3) as the template;

[0016] (5) Library construction of the amplification product of step (4), sequencing analysis, and integration site of lentiviral vector in CAR-T cells.

[0017] The existing Alu-gag PCR technology differs from the present application in that only the sequence between the Alu sequence and the gag is amplified when pre-amplification is performed, and there is no comparison of the capture efficiency of Alu and other anchor sequences of HIV. The present application finds that the capture efficiency is relatively low by comparing three different HIV anchor sequences. In the analysis method of the integration site of the lentiviral vector in the CAR-T cell, although the method can well capture the integrated HIV LTR region and part of the host genome sequence, due to the limitation of the streptomycin magnetic bead collection amplification product technology, only a few hundred bp size of the integrated sequence can be captured, and the read length is short; and the operation steps of the method are complicated, the cost of streptomycin magnetic beads is high, and the method is not suitable for rapid detection.

[0018] Therefore, how to provide a kit for rapidly and quantitatively detecting integrated HIV provirus genome is still a technical problem to be solved by those skilled in the art. SUMMARY

[0019] Based on the above reasons, the present application provides a kit for rapidly and quantitatively detecting integrated HIV provirus genome and its application, to rapidly, efficiently and at low cost solve the detection of integrated HIV and obtain the integration site information of HIV in the genome, so as to complete the quantification and chromosomal localization of the integrated HIV genome. In order to achieve the purpose of the present application, the following technical solutions are adopted:

[0020] The present application relates to a kit for rapidly and quantitatively detecting integrated HIV provirus genome and a method for rapidly locating the integration site of HIV. The kit comprises anchor primers in the Alu region of HIV virus and degenerate primers in the gag, nef and LTR regions of HIV sequence. The kit has high sensitivity (5-10 copy number / μl), which provides a technical guarantee for accurately evaluating the treatment effect of HAART and accurately quantifying the virus reservoir in HIV patients

[0021] In a preferred embodiment of the present application, the kit further comprises TaqMan probes matched with the degenerate primers in the gag, nef and LTR regions of HIV sequence.

[0022] In a preferred embodiment of the present application, the kit comprises the following primers:

[0023]

[0024] The preferred primers and probes of the present application have high genotype coverage, high conservation and strong specificity. Tests show that the kit of the present application can cover various genotypes of HIV-1, such as B, 01-AE, 07-BC, 08-BC, etc.

[0025] In a preferred embodiment of the present application, the kit further comprises fluorescent quantitative PCR primers, the primer sequence is:

[0026]

[0027] The real-time amplification curve of the amplification using the above fluorescent quantitative PCR primers has a linear relationship between the PCR cycle number and the copy number of the HIV integration standard, the Ct value of the 10X diluted standard strictly reacts 3.3 cycles of difference, and when the standard concentration is 10 1 copies / μl-10 5 copies / μl, the amplification curve shows a gradient distribution. After linear regression analysis of the data, the standard curve is obtained, and the measured values are basically on a straight line, which has good correlation with the corresponding Ct value (correlation coefficient R2=0.999), which fully illustrates that the method has high sensitivity, specificity and stability. In the repeatability experiment, the coefficient of variation (CV) is less than 2%, the error is small, and it is indicated that the kit of the present application has good stability.

[0028] In a preferred embodiment of the present application, the kit further comprises a plasmid HIV-NL4-EGFP-CMV-mCherry for packaging pseudovirus, the plasmid comprises HIV promoter, EGFP, promoter CMV and mCherry sequence, wherein the EGFP is expressed by the promoter of HIV, and the mCherry is expressed by the promoter CMV.

[0029] In a preferred embodiment of the present application, the map of the plasmid HIV-NL4-EGFP-CMV-mCherry for packaging pseudovirus is as shown in Figure 2 .

[0030] In a preferred scheme of the present application, the HIV integration standard is Jurkat-mCherry single positive cell genomic DNA quantified by a plasmid with a known copy number.

[0031] In a preferred scheme of the present application, after determining the HIV copy number of the Jurkat-mCherry single positive cell genomic DNA, gradient dilution is carried out and pre-amplification is carried out simultaneously with the sample to be tested, and the genomic DNA after pre-amplification is used as a standard for quantifying the HIV integration copy number of the sample to be tested.

[0032] Another aspect of the present application also relates to the use of the above-mentioned kit, and the kit is used for preparing an HIV virus detection reagent.

[0033] In a preferred embodiment of the present application, the kit is used for detecting integrated HIV-1 DNA in virus-infected cells.

[0034] Beneficial effects

[0035] The kit of the present application can detect the integrated copy number of QPCR after pre-amplification, which is obviously higher than that of Alu-gag and Alu-nef, and has higher capture efficiency. At the same time, on the basis of Alu-LTR capture and detection of integrated copy number, the kit of the present application can perform three-generation sequencing on samples with higher integrated level, so as to analyze the sequence composition and genomic position of HIV "reservoir". BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Schematic diagram of HIV-NL4-EGFP-CMV-mCherry double-label construction;

[0037] Figure 2 Plasmid map of HIV-NL4-EGFP-CMV-mCherry double-label;

[0038] Figure 3 Fluorescence observation after transfection of 293T cells with HIV-NL4-EGFP-CMV-mCherry double-label plasmid for 48h: (A) e-GFP fluorescence after transfection, (B) CMV-mcherry fluorescence of Jurkat cells 48h after transfection: (C) e-GFP fluorescence after lentivirus transduction, (D) CMV-mcherry fluorescence after lentivirus transduction;

[0039] Figure 4 Flow cytometry sorting of Jurkat cells 48h after transduction with the packaged pseudovirus in Example 2: (A) live cell gating; (B) single cell gating (C) mCherry and e-GFP double positive cell gating and collection;

[0040] Figure 5 Amplification curve, wherein A: Nested-QPCR HIV integration real-time amplification curve; B: HIV integration standard curve;

[0041] Figure 6 Detection of integrated HIV-1 DNA and non-integrated HIV-1 DNA copy number of four HIV-positive patients with gag as anchor primer: A: real-time amplification curve of first-round amplification of Alu-gag and sample with only gag primer, and second-round Nested-QPCR; B: electrophoresis map of first-round amplification product of Alu-gag; C: copy number of first-round amplification of Alu-gag and sample with only gag primer, and second-round Nested-QPCR;

[0042] Figure 7Detection of integrated and non-integrated HIV-1 DNA copy number in four HIV positive patients using Nef as anchor primer; A: Real-time amplification curves of the first round amplification of Alu-nef with both primers and only nef primer samples with the second round Nested-QPCR; B: Electrophoresis map of the first round amplification of Alu-nef; C: Copy number of the first round amplification of Alu-nef with both primers and only nef primer samples with the second round Nested-QPCR;

[0043] Figure 8 Detection of integrated and non-integrated HIV-1 DNA copy number in four HIV positive patients using LTR as anchor primer; A: Real-time amplification curves of the first round amplification of Alu-LTR with both primers and only LTR primer samples with the second round Nested-QPCR; B: Electrophoresis map of the first round amplification of Alu-LTR; C: Copy number of the first round amplification of Alu-LTR with both primers and only LTR primer samples with the second round Nested-QPCR;

[0044] Figure 9 A: Comparison of the first round amplification curves of three different detection methods; B: Comparison of integrated HIV DNA copy number of integrated level based on Alu-LTR, Alu-gag and Alu-nef, each black dot represents integrated HIV DNA copy number of each HIV infected person based on different detection methods;

[0045] Figure 10 Copy number of Total HIV DNA and integrated HIV DNA in brain tissue samples of HIV-1 infected persons;

[0046] Figure 11 Copy number of Total HIV DNA (A) and integrated HIV DNA (B) in PBMC samples of HIV-1 infected persons;

[0047] Figure 12 Distribution characteristics of HIV integration sites in chromosomes of four HIV positive patients;

[0048] Figure 13 Classification of genes on which HIV integration occurs in the genomes of four HIV positive patients. DETAILED DESCRIPTION

[0049] In order to further understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0050] Unless otherwise specified, the reagents involved in the embodiments of the present application are all commercially available products, which can be purchased through commercial channels.

[0051] Example 1: Preparation of HIV integration standard

[0052] The specific method for preparing and quantifying the standard for integrated HIV genome is as follows: constructing a double-tag HIV NL4-3 plasmid with defective envelope gp120, co-transfecting a vesicular stomatitis virus (VSV-G) envelope plasmid to package a pseudovirus that can only perform one round of infection, using the pseudovirus to infect human lymphocyte leukemia Jurkat cells, reducing the non-integrated HIV genome to an extremely low copy or even non-existent after 20 generations of in vitro cell passage, extracting total DNA of cells containing integrated HIV genome, and then preparing the standard for integrated HIV genome, using the plasmid DNA with known copy number diluted in gradient as a template to absolutely quantify the integrated HIV genome standard, thereby determining the integrated HIV copy number, which is used to establish the subsequent integration standard.

[0053] 1. Construction and verification of double-tag HIV-NL4-3 plasmid with defective envelope gp120

[0054] The sequence between vpu and RRE of HIV-NL4-3-EGFP plasmid is deleted, so that the plasmid cannot normally express the envelope of HIV virus.

[0055] The specific steps are: HIV-NL4-3-EGFP plasmid is double enzyme cut with EcoR I and NgoM IV restriction endonuclease, the product is subjected to 0.8% agarose gel electrophoresis, and the large fragment is recovered in an Eppendorf tube, the corresponding fragment is recovered by using the agarose gel recovery kit of TIANGEN company, and the purity and concentration of the product are determined. The recovered fragment of the above-mentioned vector is added with a linker sequence, a CMV promoter and a CMV enhancer, and a gel recovery product of the mCherry fragment in an equal molar ratio into an Eppendorf tube, Exnase II ligase and homologous recombinase 5x CE II buffer are added, and the reaction is carried out at 37°C for 0.5 hours; 10 μL of the homologous recombination product is taken out, 100 μL of Stabl3 competent cells are added, ice bath is carried out for 30 min, and then 42°C heat shock is carried out for 90 s, 500 μL of SOC medium is added, and the culture is carried out at 37°C and 220 rpm for 2 hours; after 2 hours, centrifugation is carried out at 4000 g for 1 min, and 400 μL of excess liquid is removed. The remaining liquid is spread on an LB plate containing ampicillin and cultured at 37°C for 12 hours; single colonies are picked up on the plate, respectively, and inoculated into 5 mL of LB liquid medium, and cultured at 37°C and 220 rpm for 12 hours. The plasmid is extracted by using the small extraction kit of TIANGEN company, the plasmid HIV-NL4-EGFP-CMV-mCherry plasmid is obtained, and after the sequencing verification of Shengong Biotechnology (Shanghai) Co., Ltd. Science and Technology Company shows no error, the Stabl3 strain containing the HIV-NL4-EGFP-CMV-mCherry plasmid is preserved. The schematic diagram of HIV-NL4-EGFP-CMV-mCherry plasmid construction is shown in Figure 1 The complete map of HIV-NL4-EGFP-CMV-mCherry plasmid is shown in Figure 2 .

[0056] Example 2: Packaging and integration of HIV-NL4-EGFP-CMV-mCherry pseudovirus and establishment of HIV stable cell line

[0057] The double-tagged HIV NL4-3 plasmid with envelope gp120 defect is co-transfected with the vesicular stomatitis virus (VSV-G) envelope plasmid into 293T cells to package the pseudovirus. The pseudovirus is used to infect Jurkat cells. Since EGFP is expressed by the HIV promoter, and CMV-mCherry is expressed by the strong CMV promoter, its expression is not interfered by the HIV promoter. When the pseudovirus infects Jurkat cells and the number of passages increases, HIV will gradually integrate and hide in the genome of the cells. At this time, the Jurkat cells only express mCherry, so the Jurkat-mCherry cells obtained by flow sorting can be used as an HIV-integrated cell line. The total DNA of the cells is extracted and prepared for the standard of integrated HIV genome.

[0058] 1. Pseudovirus packaging

[0059] Logarithmic growth phase 293T cells (5 x 106 / 10 cm dish), 10 dishes, the cells were inoculated into the dish, Flask 24 h before transfection, the culture conditions were DMEM + 10% FBS, 37℃, 5% CO2; according to the volume of lentivirus preparation and system requirements, the amount of plasmid, packaging plasmid, PEI was calculated. Plasmid VSVG = 5:2, 10 μg of plasmid and 30 μL of PEI were added to each 10 cm dish. Before transfection, 2 50 ml centrifuge tubes were taken, and 16 mL (500 μL) of DMEM base medium (without FBS) was added to each tube. One tube was added with 960 μg (30 μg) of PEI, and the other tube was added with 320 μg (10 μg) of premixed plasmid (plasmid VSVG = 5:2). Vortex for 5 min, and then incubate at room temperature for 30 min. According to the amount of 1 ml per 10 cm dish, the PEI and plasmid mixed DMEM base medium was added to the 10 cm dish of 293T cells, and the 10 cm culture dish was placed in a 37℃ 5% CO2 incubator for 72 h. The supernatant virus stock was collected.

[0060] 2. Establishment of integrated HIV double-tagged genome stable cell line

[0061] Jurkat cell suspension was taken 10ul count, according to the count, to determine the volume of resuspension. Take the appropriate volume of cell suspension, put the cells into the centrifuge, at room temperature 300g, centrifuged for 5 minutes, discard the supernatant. Determine the transduction MOI, add the lentivirus, add 1640 serum-free medium to resuspend to 1ml, add transfection reagent Polybrene to a final concentration of 8ug / ml, inoculate into a 12-well plate, incubate for about 4h. After incubation, 300g centrifugation for 5min, make a mark, remove the supernatant, resuspend to 2x10 6 cells / ml, add to a six-well plate, put into a 37.0℃, 5.0% carbon dioxide incubator for 24h, and preliminarily observe the fluorescence. When the cell density reaches about 3x10 6 / ml, it needs to be passaged. At 72h, Jurkat-mCherry single positive cells were sorted by flow cytometry, and the single positive cells were cultured to expand as HIV integration standards.

[0062] Example 3: Establishment of HIV-1 integration standard curve

[0063] Jurkat-mCherry cells in Example 2 as HIV latent infection cell line can be used to quantify the HIV integration standard of clinical HIV infected patients.

[0064] 1. Expansion culture of Jurkat-mCherry cells

[0065] Jurkat-mCherry cells obtained by flow sorting in Example 2 were expanded to 1x10 7 , and washed with PBS three times, and finally resuspended in 10ul complete medium to 1.5ml centrifuge tube.

[0066] 2. DNA extraction of Jurkat-mCherry cells

[0067] Take 1x10 7PBMC was added 1 mL DNAZOL, gently blow about 50 times, pay attention to avoid bubbles. To the cell homogenate liquid added 0.5 mL anhydrous ethanol, upside down 10 times, room temperature for 1-3 min, visible white DNA precipitation; 4℃, 18000g, centrifugal 15 min; remove supernatant, add 1 mL 75% ethanol solution washing, upside down 5 times, room temperature for 1-3 min; 4℃, 15000g, centrifugal 5 min; remove supernatant, open cover and stand to make the precipitation air dry; each sample was added 100 μl of 8 mM NaOH solution, 4℃ dissolved overnight; each sample was added 8.6 μl 0.1 M HEPES, adjust pH to 8.4. The DNA concentration A260nm / A230nm, A260nm / A280nm was determined by ultramicro UV spectrophotometer Q5000.

[0068] 3. Absolute quantification of Jurkat-mCherry cell genomic DNA

[0069] Select HIV NL4-3 as template, primer HIV-112.0 to amplify HIV-1 target gene and calculate plasmid copy number according to the following formula: plasmid copy number = (plasmid concentration x plasmid volume / plasmid relative molecular mass) x 6.02 x 10 23 . 10-fold dilution of known concentration of plasmid standard to make its linear concentration range 10 1 copies / μl ~ 10 6 copies / μl. Quantification of Jurkat-mCherry cell DNA HIV copy number.

[0070] Table 1: QPCR reaction system

[0071]

[0072] Open Bio-Rad PrimePCR fluorescence quantitative PCR instrument, set the program according to the following table, put in eight joint tube, detect on the same plate and read the copy number of integrated HIV

[0073] According to the measured copy number of Jurkat-mCherry cell DNA as integrated HIV genome standard, the concentration linear range of the standard was adjusted to 10 1 -10 5 copy number / μl, DNA concentration of 10 5 copy number / μl standard was 10-fold serial dilution, and uninfected cell DNA was added. The purpose of adding uninfected cell DNA was to make the concentration of cell DNA in each concentration of HIV integration quantitative standard be 10 5The copy number / μl corresponds to the same DNA concentration in the standard sample, ensuring that the amplification efficiency of the downstream integrated HIV DNA in the pre-amplification process is consistent.

[0074] 4. Establishment of integrated HIV standard curve

[0075] The nested fluorescent quantitative PCR method of the present application quantifies integrated HIV in two steps:

[0076] Considering the structural characteristics of HIV virus integrated into the host cell genome, the downstream and upstream anchor primers are designed on the highly conserved LTR, Gag, and nef regions of the HIV virus genome sequence and the Alu repeat sequence of the human genome, respectively. The pair of primers can specifically capture the integrated HIV genome sequence (containing the 5' LTR part) and linearly amplify the copy number of the amplicon after 12 cycles of pre-amplification, greatly improving the specificity and sensitivity of quantification.

[0077] In the 5' LTR region of HIV included in the pre-amplification PCR, a pair of primers and a matching TaqMan probe are designed. The first round of amplification standard and the amplification product are used as the second round of fluorescent quantitative PCR standard and the sample to be tested, respectively. The second round of fluorescent quantitative PCR is performed. Compared with the standard curve, the absolute copy number of the sample to be tested is read out. The copy number of the integrated HIV provirus of the sample to be tested is equal to the difference between the copy number of the sample with Alu primer and the copy number of the reference sample without Alu primer.

[0078] (1) 12 cycles of pre-amplification

[0079] 10-fold gradient dilution of known copy number of HIV integration standard sample to make its linear concentration range 10 1 copies / μl-10 5 copies / μl. Pre-amplification is performed simultaneously with the sample to be tested, and the reaction system is 25 μl.

[0080] Table 2: First round of pre-amplification PCR reaction system

[0081]

[0082] Mix thoroughly and centrifuge briefly, then prepare for machine operation BioRad PCR instrument, and the amplification program is as follows:

[0083] Table 3: First round of pre-amplification PCR reaction program

[0084]

[0085] Note: 2x Phanta Max Master Mix is a high-fidelity super-long fragment Taq DNA polymerase.

[0086] Table 4: Integration site capture primers

[0087]

[0088]

[0089] (2) Second round of fluorescent quantitative PCR amplification

[0090] The first round of pre-amplified standards and amplification products were centrifuged and used as the second round of fluorescent quantitative PCR standards and samples to be tested. The preparation of the fluorescent quantitative PCR reaction solution is shown in Table 1: the reaction primers are shown in Table 5 below

[0091] Table 5: Second round of fluorescent quantitative PCR primer sequences

[0092]

[0093] (3) Nesting fluorescent quantitative PCR preparation amplification curve and standard curve generation

[0094] In the present application, the amplification reaction uses Bio-Rad real-time fluorescent quantitative PCR instrument, and the amplification program is shown in Table 6:

[0095] Table 6: Second round of fluorescent quantitative PCR amplification program

[0096]

[0097] (4) Detection channel selection: fluorescent channel selection FAM.

[0098] The copy number of the HIV integration standard added during pre-amplification was input into the Bio-Rad analysis software to obtain the real-time amplification curve of each standard tube (as shown in Figure 3 ), and further using the logarithmic value of the copy number of the HIV integration standard added during pre-amplification as the abscissa and the Ct value as the ordinate, a standard curve was drawn (as shown in Figure 5 ). There is a linear relationship between the number of PCR cycles in the real-time amplification curve of the fluorescent quantitative PCR and the copy number of the HIV integration standard, and the Ct value of the 10X diluted standard strictly follows the difference of 3.3 cycles, and when the standard concentration is 10 1 copies / μl-10 5 copies / μl, the amplification curve shows a gradient distribution. After linear regression analysis of the data, a standard curve was obtained, and the measured values were basically on a straight line, and had good correlation with the corresponding Ct value (correlation coefficient R 2 = 0.999), fully demonstrating the high sensitivity, specificity and stability of the method. In the repeatability experiment, the coefficient of variation (CV) was <2%, the error was small, and it was indicated that the detection method had good stability.

[0099] When detecting integrated HIV-1 virus genome in the sample to be tested, the HIV integration standard and the sample to be tested are prepared at the same time, the sample to be tested without Alu primer is used as a reference sample, the above-mentioned nested fluorescent quantitative PCR operation is carried out, the absolute copy number of the sample to be tested and the reference sample is read by comparing the sample to be tested and the reference sample with the standard curve prepared by the nested fluorescent quantitative PCR, and the copy number of the integrated HIV provirus in the sample to be tested is equal to the difference between the copy number of the sample with Alu primer and the copy number of the reference sample without Alu primer.

[0100] Example 4: Capture of integration site in PBMC of HIV positive infected person and quantitative determination of integrated HIV by nested-QPCR

[0101] 1. Extraction of genomic DNA from PBMC (human peripheral blood lymphocytes) of HIV positive infected person

[0102] (1) Sample homogenization

[0103] Take 1x107PBMC and add 1mL DNAZOL, gently blow about 50 times, and avoid bubbles.

[0104] (2) DNA precipitation

[0105] Add 0.5mL anhydrous ethanol to the above cell homogenate, invert 10 times, and stand at room temperature for 1-3min, and white DNA precipitate can be seen; centrifuge at 4℃, 18000g, for 15min;

[0106] (3) DNA washing

[0107] Remove the supernatant, add 1mL 75% ethanol solution for washing, invert 5 times, stand at room temperature for 1-3min; centrifuge at 4℃, 15000g, for 5min; remove the supernatant, open the cover and let the precipitate air dry;

[0108] (4) DNA dissolution

[0109] Add 100μl of 8mM NaOH solution to each sample, and dissolve at 4℃ overnight; add 8.6μl of 0.1M HEPES to each sample to adjust the pH to 8.4.

[0110] (5) DNA concentration determination

[0111] Determine the DNA concentration A260nm / A230nm, A260nm / A280nm, etc. by using ultramicro UV spectrophotometer Q5000.

[0112] 2. PCR capture of integration site Alu-gag and Alu-nef

[0113] (1) Design primers for gag and nef of high-conserved region of domestic epidemic strain and primers for conserved region of Alu sequence, and capture Alu-gag and nef-Alu integration sites respectively. The reaction system is shown in Table 2. The primers used in the capture are shown in Table 5.

[0114] (2) PCR product purification: add equal volume of phenol: chloroform: isopentanol (25:24:1), invert several times, centrifuge at 14000 rpm for 10 min at 4℃;

[0115] (3) Transfer the supernatant (aqueous phase) to a new 1.5 ml EP tube; add 1 / 10 volume of 3M NaAc (pH 5.2), invert several times to mix well; then add twice volume of ice ethanol, mix again, and precipitate at -20℃ overnight; the next day, centrifuge at 14000 rpm for 10 min at 4℃;

[0116] (4) Wash the DNA precipitate with 75% ethanol twice, and place in air to evaporate the residual liquid; add appropriate volume of water to dissolve the DNA precipitate, and after complete dissolution, measure the concentration with a nucleic acid quantifier and perform electrophoresis detection.

[0117] 3. Quantification of integrated HIV based on nested-QPCR of Alu-gag and Alu-nef pre-amplification

[0118] Prepare and add samples according to the primers and probes and reaction system of the fluorescence quantitative reaction in Example 3, using the PCR capture product of the previous step as the template, to quantify the integrated HIV of the following HIV-positive infected persons.

[0119] Open the Bio-Rad PrimePCR fluorescence quantitative PCR instrument, set the program according to the following table, and place it in an eight-tube tube for detection on the same plate.

[0120] Reaction temperature and time: 95℃ for 5 min, 1 cycle; finally 94℃ for 10 s, 60℃ for 30 s, 40 cycles to collect fluorescence signal. The results of integration quantification based on Alu-gag are shown in Figure 6 , and the results of integration quantification based on Alu-Nef are shown in Figure 7 .

[0121] Example 4: Optimization of HIV integration site capture system

[0122] 1. Quantification of integrated HIV DNA copy number based on Alu-LTR capture

[0123] Considering the limitations of Alu-gag PCR for detecting integrated HIV, including the distance of HIV-1 integration site to the nearby Alu sequence which can affect the efficiency of PCR, the genomic DNA of HIV positive infected PBMCs was selected for Alu-LTR amplification, i.e. the Alu primer remained unchanged, and the gag primer was replaced by a primer for the 5' LTR region of HIV long terminal repeat sequence closer to the Alu sequence. The PCR product was then detected for integrated HIV according to the primers in Example 3. In general, the results of integrated HIV captured by Alu-LTR were better than those captured by Alu-gag and Alu-nef.

[0124] 2. Comparison of three methods for detecting integrated HIV DNA copy number by Alu-LTR, Alu-gag and Alu-nef

[0125] In order to verify whether the method of capturing HIV integration site by Alu-LTR is better than that by Alu-gag and Alu-nef, the copy number of integrated HIV detected by the three methods was compared, which was normalized to the copy number per million PBMC cells. Considering individual differences, the Nested-QPCR detection of integrated HIV based on Alu-LTR, Alu-gag and Alu-nef showed higher detection efficiency in WFQ patients, but not in other patients. Among the three detection methods, the detection of integrated HIV based on Alu-LTR was significantly better than that based on Alu-gag and Alu-nef. Therefore, the capture product of Alu-LTR was selected for three-generation sequencing, and the next step of analysis of HIV integration site was carried out. Here, a detection method using nef and LTR as HIV anchor targets was invented, and it has been proved that the primer combination of the present invention improves the quantification of integrated HIV-1 DNA copies compared with any method used before.

[0126] Example 5: Quantitative detection of HIV integrated provirus in brain tissue samples of HIV-1 infected patients

[0127] 1. Sample processing (in a BSL-2+ laboratory)

[0128] After the hospital ethics committee, obtain 14 cases of HIV-1 infected brain tissue samples and blood samples, cut 1mg or so gray matter or white matter into 200ul Trizol (Invitrogen) and grind thoroughly, then transfer 100ul grinding mixture to 900ul DNAzol, blow thoroughly, then add 500ul anhydrous ethanol, centrifuge at 14000rpm for 15 minutes, wash twice with 1ml 75% anhydrous ethanol, add 8mM NaOH, store at 4℃ overnight, then adjust pH to 8.5 with 1M HEPES buffer and store at -80℃.

[0129] 2. Nested-QPCR detection of integrated HIV provirus in brain tissue samples of HIV-1 infected patients

[0130] According to the method of step 1 in Example 3 of the present application, the total HIV DNA in the sample to be tested is quantified, and according to the experiment and method of step 4 of Example 3, the integrated HIV provirus is quantified. When quantifying the integrated HIV provirus, all samples to be tested will set a parallel amplification tube without Alu primer as a reference sample according to the method of Example 4. The detection data are shown in Table 7 and Figure 10

[0131] Table 7 Integrated HIV DNA and total HIV DNA detected in brain tissue of HIV-1 infected patients

[0132] Sample Total DNA copy number Integrated DNA copy number Total copy number / integrated copy number 531g 73.53 15.00 4.90 500g 306817.67 5271.51 58.20 537w 107882.66 8707.87 12.40 537g 405229.30 26838.82 15.10 017w 84744.22 683.10 124.10 017g 187315.73 10058.21 18.60 070w 192800.11 9006.73 21.40 070g 85250.47 27336.29 3.10 013g 9400.64 888.00 10.59 509w 352.56 9.70 36.30 509g 329.36 14.00 23.53 133w 3429.26 191.57 17.90 133g 140.95 46.54 3.00 103w 59517.61 14168.14 4.20

[0133] According to the above data, the average value of the ratio of the copy number of total HIV-1 DNA to the copy number of integrated HIV-1 DNA in the brain tissue sample is 25, i.e. the copy number of total HIV-1 DNA is 25 times the copy number of integrated HIV-1 DNA in the brain tissue sample. The detection results show that HIV-1 DNA mainly exists in the form of non-integration in brain tissue cells. If the copy number of total HIV-1 DNA is used instead of the copy number of integrated HIV-1 DNA as a reference to evaluate the viral "reservoir" in the patient's body, it is inaccurate. The result analysis highlights the importance and necessity of establishing nested fluorescent quantitative PCR to accurately quantify the copy number of integrated provirus, which can be used to judge whether the brain is infected and the treatment effect after drug use, and can also determine whether HIV-1 establishes a viral reservoir in the brain and plays an important role in further studying the relationship between the reservoir and HIV-1 induced nerve damage.

[0134] 3. Nested-QPCR detection of integrated HIV provirus in PBMC of HIV-1 infected patients

[0135] ​The total HIV DNA in the sample to be tested is quantified according to the method of step 1 in Example 3, the integrated HIV provirus is quantified according to the experiment and method of step 4 of Example 3, and when the integrated HIV provirus is quantified, all samples to be tested are set with a parallel amplification tube without Alu primers as a reference sample according to the method of Example 4. The detection data are shown in Table 7 and Figure 11

[0136] Example 6: HIV integration site sequencing and bioinformatics analysis

[0137] 1. Preparation of Alu-LTR capture product

[0138] The PCR product in step 4 of Example 3 is sent to Wuhan Benya Biotechnology Co., Ltd. for sequencing using the Nanodrop platform, and bioinformatics analysis is performed on the data obtained by high-throughput sequencing.

[0139] 2. Construction of PCR product library

[0140] The PCR fragment to be tested is end-repaired and repaired, and a base "A" is added at the 3' end to convert it into a sticky end. Then a DNA adapter is added on both sides of the sticky end through base complementarity. PCR amplification is performed to add Index at the end of the target fragment, and the construction and quality inspection of the sequencing library are completed.

[0141] 3. Sequencing

[0142] The sequencing library is combined with the sequencing chip by bridge PCR, and sequencing is performed.

[0143] 4. Bioinformatics analysis

[0144] After obtaining the raw data with sufficient sequencing depth, bioinformatics analysis is started, which mainly includes the following three stages: sequencing data evaluation: the sequencing quality evaluation is performed to evaluate the data quality to meet the analysis requirements, the quality-controlled data is used for comparison, and the data quality is evaluated to meet the requirements for the next step analysis; the readl and read2 of Cleanreads are combined according to the overlap sequence to generate single reads, and the combined reads are compared with the genome of human and HIV, and the comparison results are counted; the integration site is detected according to the comparison results, and the integration site is counted and analyzed.

[0145] The above describes the preferred embodiments of the present application, but is not intended to limit the present application. Those skilled in the art can make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present application.​

Claims

1. A kit for quantitatively detecting integrated HIV proviral genomes, comprising an anchor primer in the Alu region of HIV virus and degenerate primers in the gag, nef and LTR regions of HIV sequences.

2. The kit according to claim 1, further comprising TaqMan probes that match degenerate primers in the gag, nef and LTR regions of HIV sequences.

3. The kit according to claim 1, comprising the following primers:

4. The kit according to claim 1, further comprising fluorescent quantitative PCR primers, wherein the primer sequences are:

5. The kit according to claim 1, further comprising an HIV integration standard plasmid, wherein the plasmid comprises an HIV promoter, EGFP, a CMV promoter, and an mCherry sequence, wherein EGFP is expressed by the HIV promoter, and mCherry is expressed by the CMV promoter.

6. The kit according to claim 5, wherein the map of the HIV integration standard plasmid is shown in FIG2.

7. Use of the kit according to any one of claims 1 to 6, wherein the kit is used for preparing an HIV virus detection reagent.

8. The use according to claim 7, wherein the kit is used for detecting integrated HIV-1 DNA in virus-infected cells.

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