A method for detecting nucleic acid integrity based on digital PCR

Abstract

The present invention is based on a method for detecting nucleic acid integrity by six-channel digital PCR. The kit used includes a plurality of primers and probes containing 1-3 degenerate bases, which can identify and amplify multiple similar fragments of target nucleic acid. At the same time, the 5' end of most probes is labeled with a fluorescent dye that is different from the 5' end of other probes. After digital PCR amplification, droplets containing fluorescent signals represented by different colors released by different fluorescent dyes are obtained. After that, the droplets containing different fluorescent signal colors in a digital PCR reaction well are analyzed and interpreted to distinguish between intact and defective nucleic acids, which can improve the detection accuracy. This method overcomes the phenomenon that fluorescent labels are limited in existing digital PCR technology, two reaction wells are required for detection, and detection information is missing.

Description

Technical Field

[0001] The present application relates to the field of gene detection technology, and in particular to a method for detecting nucleic acid integrity based on digital PCR for non-diagnostic purposes. Background Art

[0002] The inability to eradicate the human immunodeficiency virus (HIV, the main prevalent strain is HIV-1) in the body is the fundamental reason why AIDS patients are difficult to cure. Studies have shown that this phenomenon occurs because HIV-1 nucleic acid will stably integrate into CD4 + On the genome of T cells, these latently infected CD4 + Although T cells do not express viral proteins, they can exist stably in the infected body for a long time, thus forming a stable latent reservoir and evading the surveillance and elimination of the host immune system. + T cells carry nucleic acid of the integrated HIV-1 provirus, and some are integrated into CD4 + HIV-1 proviral nucleic acids on T cells may contain large internal deletions or APOBEC-3G-induced hypermutations or other inactivating mutations, such as packaging signal deletions, insertions, deletions, and missense mutations of small nucleotide fragments. These CD4 T cells containing incomplete HIV-1 nucleic acids may be infected with HIV-1. + T cells do not have the ability to express viral proteins and are therefore usually defined as inactive nucleic acids. Only those containing intact HIV-1 nucleic acids are active. Therefore, accurately evaluating and measuring the amount of intact HIV-1 nucleic acids in HIV-1 latent reservoirs is an important indicator of treatment prognosis.

[0003] At present, there are mainly several detection methods for detecting HIV-1 nucleic acid integrity: the first is the Viral Outgrowth Assay (VOA), which is the "gold standard" detection method. This detection method requires 120 to 180 mL of blood for each test, and requires cell culture for 1 to 3 weeks in a biosafety level 3 laboratory to induce HIV-1 to be released from the latent reservoir. Therefore, it has the defects of long detection cycle and underestimation of nucleic acid integrity; the second method is the Alu-PCR method, which requires two rounds of PCR process and cannot distinguish between defective and replication-competent proviruses; the third method is the digital PCR method (digital PCR, dPCR). The main principle of digital PCR technology is to randomly disperse the sample into thousands of tiny reaction units of equal volume (also called droplets), and then perform PCR amplification on the reaction units. After the amplification, the fluorescence signal of each reaction unit is read and analyzed. This technology can accurately detect low-abundance targets, targets in complex mixtures, and small multiple differences of target genes, and can be applied to a wide range of sample types and application fields. The complete proviral nucleic acid detection method (Intact Proviral DNA Assay, IPDA method) based on digital PCR technology is based on the fact that 90% of defective viruses have defects in the packaging signal segment (Ψ segment) and env segment of HIV-1 nucleic acid, and detects the Ψ segment and env segment based on a dual-channel digital PCR platform to identify complete viruses or defective viruses. However, subsequent studies have shown that the env segment will have polymorphic variations, so this method is difficult to obtain comprehensive HIV-1 nucleic acid variation characteristics, and each detection requires two reaction wells (one reaction well detects the Ψ segment and env segment of HIV-1 nucleic acid; the other reaction well is used to detect the internal standard gene to obtain the shear coefficient of the nucleic acid to correct the integrity of the original virus). Such an operation not only increases the consumption of samples, but also increases the detection cost. In addition, this method cannot detect the original virus whose defects occur outside the packaging signal ψ segment and env segment, resulting in missing detection information. At present, there is still a lack of a comprehensive, accurate, quantitative, low-sample usage, and low-testing cost detection method for HIV-1 nucleic acid integrity in a single digital PCR reaction well at home and abroad.

[0004] In summary, there is an urgent need to develop a method for detecting HIV-1 nucleic acid integrity that is simple, comprehensive, accurate and quantitative in a single digital PCR reaction well, can reduce sample usage and reduce testing costs. Summary of the invention

[0005] Based on this, it is necessary to provide a method for detecting HIV-1 nucleic acid integrity that is simple, comprehensive, accurate and quantitative in a single digital PCR reaction well, can reduce the amount of sample used and reduce the cost of detection. Of course, the method of this application can be used not only for the detection of HIV-1 nucleic acid integrity, but also for the detection of nucleic acid integrity of other species.

[0006] The present application provides a method for detecting nucleic acid integrity based on digital PCR for non-diagnostic purposes, comprising the following steps:

[0007] A digital PCR reaction system for detecting a sample is prepared using a kit, and the sample is added; wherein the kit includes a plurality of primers and a plurality of probes, and at least some of the primers and probes contain 1-3 degenerate bases; the 5' ends of the plurality of probes are labeled with a fluorescent dye that is different from the 5' ends of other probes, so that after the plurality of probes are amplified by PCR in the same digital PCR reaction well, the amplification products can be distinguished by positive droplets that generate different types of fluorescent signals;

[0008] Loading the digital PCR reaction system into the digital PCR chip;

[0009] The digital PCR chip is placed in a digital PCR instrument for PCR amplification to obtain an amplification result; the digital PCR instrument is a Naica six-channel droplet chip digital PCR instrument.

[0010] The digital PCR chip having the amplification result is placed in a chip scanner to scan the fluorescence signal of the amplified droplets, and the positive droplets containing fluorescent signals of different colors are subjected to fluorescent crosstalk analysis, and then the nucleic acid integrity is judged according to the positive droplets containing different fluorescent signal colors after the analysis; wherein the corresponding relationship between the positive droplets containing different fluorescent signal colors and the nucleic acid integrity is shown in Table 1;

[0011] Table 1. Correspondence between droplets with different fluorescent signal colors and nucleic acid integrity

[0012]

[0013] In one embodiment, the 5' end of the probe is connected to a fluorescent reporter group, and the 3' end is connected to a fluorescent quencher group.

[0014] The above-mentioned method for detecting nucleic acid integrity based on six-channel digital PCR uses a kit including multiple primers and probes containing 1-3 degenerate bases, which can identify and amplify multiple similar fragments of the target nucleic acid. At the same time, the 5' end of most probes is labeled with a fluorescent dye that is different from the 5' end of other probes. After digital PCR amplification, droplets containing fluorescent signals represented by different colors released by different fluorescent dyes are obtained. Then, the droplets containing different fluorescent signal colors in a digital PCR reaction well are analyzed and interpreted to distinguish between complete and defective nucleic acids, which can improve the detection accuracy. This method can overcome the phenomenon of limited fluorescent labeling in existing digital PCR technology, the need to use two reaction wells for detection, and the lack of detection information.

[0015] In one embodiment, the kit includes a plurality of forward primers containing degenerate bases, a plurality of reverse primers containing degenerate bases, and a plurality of probes containing degenerate bases.

[0016] In one embodiment, the method is used to detect intact HIV-1 nucleic acid.

[0017] In one embodiment, the method is used to detect multiple defective HIV-1 nucleic acids.

[0018] In one embodiment, the plurality of forward primer sequences include Ψ-f, pol-5'f, pol-3'f, env-1f, env-2f, RPP30-1f, RPP30-2f; the plurality of reverse primer sequences include Ψ-r, pol-5'r, pol-3'1r, pol-3'2r, env-r, RPP30-1r, RPP30-2r; the plurality of probe sequences include Ψ-p, pol-5'p, pol-3'p, env-1p, env-2p, env-3p, RPP30-1p, RPP30-2p, wherein:

[0019] The sequence of Ψ-f is: 5′-CAGGACTCGGCTTGCTGARG-3′;

[0020] The sequence of pol-5′f is: 5′-GYCCYATTGADACTGTAC-3′;

[0021] The sequence of pol-3′f is: 5′-ARGGAYTATGGAAAACA-3′;

[0022] The sequence of env-1f is: 5′-AGCAGTGGGAMTAGGA-3′;

[0023] The sequence of env-2f is: 5′-ACTATGGGCGCRGCGTC-3′;

[0024] The sequence of RPP30-1f is: 5′-CATGCTCTGTGGTATAGC-3′;

[0025] The sequence of RPP30-2f is: 5′-AGATTTGGACCTGCGAGCG-3′;

[0026] The sequence of Ψ-r is: 5′-TCTCCTTCTAGCCTCC-3′;

[0027] The sequence of pol-5′r is: 5′-ATGGCCAYTGTTTAACYT-3′;

[0028] The sequence of pol-3′1r is: 5′-GTKCCATGTTYTAATCC-3′;

[0029] The sequence of pol-3′2r is: 5′-ATTCCATGTTCTAATCT-3′;

[0030] The sequence of env-r is: 5′-TGCTGYTGCACTATRC-3′;

[0031] The sequence of RPP30-1r is: 5′-GCTGATTTTGAAGAATTTCTG-3′;

[0032] The sequence of RPP30-2r is: 5′-GAGCGGCTGTCTCCACAAGT-3′;

[0033] The sequence of Ψ-p is: 5′-TTTTGGCGTACTCACCAGT-3′;

[0034] The sequence of pol-5′p is: 5′-AAGCCAGGAATGGATGGMC-3′;

[0035] The pol-3′p sequence is: 5′-ATCCTGTCTACYTGCCACAC-3′;

[0036] The sequence of env-1p is: 5′-CCTTGGGTTCTTGGG-3′;

[0037] The sequence of env-2p is: 5′-CCTTAGGTTCTTAGGAGC-3′;

[0038] The sequence of env-3p is: 5′-CTGGCCTGTACCGTCAG-3′;

[0039] The sequence of RPP30-1p is: 5′-TGCCAGCATGATTGTCTCAGC-3′;

[0040] The sequence of RPP30-2p is: 5′-TTCTGACCTGAAGGCTCTGCGCG-3′;

[0041] In one embodiment, the kit further includes controls, including negative controls and positive controls; the negative control is nuclease-free water, the positive control is a fragmented plasmid standard, and the fragmented plasmid standard includes the following two types: HIV-1-FL plasmid and RPP30-FL plasmid are mixed and then ultrasonically treated to form a fragmented plasmid standard; preferably, the sample of the kit is selected from whole blood or peripheral blood mononuclear cell (PBMC) samples; preferably, the kit further includes a digital PCR amplification enzyme premix and a fluorescein sodium salt solution, and the digital PCR amplification enzyme premix includes dNTPs, Taq enzyme and PCR buffer.

[0042] In one embodiment, the digital PCR reaction system is 25 μL.

[0043] In one embodiment, the PCR reaction procedure corresponding to the PCR chip is as follows:

[0044] Droplet generation step: temperature 40°C;

[0045] Pre-denaturation step: temperature 95°C, time 5 minutes;

[0046] PCR reaction step: denaturation temperature 95°C for 15 seconds, annealing temperature 60°C for 30 seconds, wherein the PCR reaction step is cycled multiple times;

[0047] Pressure release step: temperature 25°C.

[0048] In one embodiment, the digital PCR chip is a Sapphire chip.

[0049] In one embodiment, Ψ-p is fluorescently labeled with 5'-FAM, 3'-MGB; pol-5'p is fluorescently labeled with 5'-TAMRA, 3'-BHQ2; pol-3'p is fluorescently labeled with 5'-Cy5.5, 3'-BHQ3; env-1p is fluorescently labeled with 5'-VIC, 3'-MGB; env-2p is fluorescently labeled with 5'-Unlabeled, 3'-MGB; env-3p is fluorescently labeled with 5'-VIC, 3'-MGB; RPP30-1p is fluorescently labeled with 5'-ROX, 3'-BHQ2; RPP30-2p is fluorescently labeled with 5'-Cy5, 3'-BHQ3.

[0050] In one embodiment, the relationship between positive droplets containing fluorescent signals of different colors and HIV-1 nucleic acid integrity is shown in Table 2.

[0051] Table 2. Relationship between positive droplets containing different color fluorescence signals and HIV-1 nucleic acid integrity

[0052]

[0053] In one embodiment, the fluorescence signals detected by the digital PCR all have corresponding fluorescence values, and a specific fluorescence threshold and fluorescence compensation parameter are substituted into the fluorescence value to obtain the number of positive droplets in each fluorescence channel and the corresponding copy number concentration of each target nucleic acid, and the shear coefficient and other data are calculated according to the following formula:

[0054]

[0055]

[0056]

[0057] In the present application, the detection method is applied to the detection of HIV-1 nucleic acid integrity, which has the following advantages: First, the present application adopts six-channel digital PCR, and PCR amplification is simultaneously performed on four segments of HIV-1 nucleic acid and two segments of internal reference genes in one digital PCR reaction well. The detection probes of the four segments of nucleic acid and the two segments of internal reference genes are labeled with different fluorescent dyes, and the amplification of multiple segments of nucleic acid will not produce cross-reactions. After that, by dividing and analyzing the droplets with different fluorescent signal colors after amplification, not only the number of intact HIV-1 nucleic acids can be accurately determined, but also the HIV-1 nucleic acid ψ segment, pol 5' end segment, pol The number of defective nucleic acids in the 3' end segment and the env segment overcomes the shortcomings of the prior art such as few defective types detected, long detection time, large sample usage, large number of detection holes, and high cost. It not only improves the detection efficiency and accuracy, but also reduces the detection cost and sample usage; secondly, the present application adds a detection method and calculation method for the internal reference gene for calculating the shear coefficient, and further calculates the number of nucleated cells, the intact HIV-1 nucleic acid load and other more detailed data for user reference by calculating the shear coefficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 A three-dimensional graph of fluorescence signals of HIV-1 nucleic acids of different types of integrity and a two-dimensional graph of fluorescence signals of the internal reference gene RPP30 in one embodiment;

[0059] Figure 2 is a schematic diagram of the HIV-1 nucleic acid structure of Example 1;

[0060] Figure 3 This is a schematic diagram of the fluorescent channel selected when scanning for intact HIV-1 nucleic acid in Example 1;

[0061] Figure 4a This is a display diagram of all droplets after scanning a single chip well when detecting intact HIV-1 nucleic acid in Example 1;

[0062] Figure 4b This is a partial droplet display diagram of the BLUE channel (blue) when detecting intact HIV-1 nucleic acid in Example 1; Figure 4c This is a partial droplet display diagram of the GREEN channel (green) when detecting intact HIV-1 nucleic acid in Example 1; Figure 4d This is a partial droplet display diagram of the INFRA-RED channel (purple) when detecting intact HIV-1 nucleic acid in Example 1; Figure 4e This is a partial droplet display diagram of the TEAL channel (light blue) when detecting intact HIV-1 nucleic acid in Example 1; Figure 4f This is a three-dimensional stereogram of the fluorescence signals of the BLUE channel (blue) + GREEN channel (green) + TEAL channel (light blue) detected by Example 1 for intact HIV-1 nucleic acid; Figure 4g This is a three-dimensional stereogram of the fluorescence signals of the BLUE channel (blue) + INFRA-RED channel (purple red) + TEAL channel (light blue) of the intact HIV-1 nucleic acid detected in Example 1;

[0063] Figure 5a This is a partial droplet display diagram of the BLUE channel (blue) when detecting HIV-1ψ segment deletion type plasmid standard in Example 2; Figure 5b This is a partial droplet display diagram of the GREEN channel (green) of Example 2 when detecting HIV-1ψ segment-deficient plasmid standard; Figure 5c This is a partial droplet display diagram of the INFRA-RED channel (purple) of Example 2 when detecting HIV-1ψ segment-deficient plasmid standard; Figure 5d This is a partial droplet display diagram of the TEAL channel (light blue) of Example 2 when detecting HIV-1ψ segment-deficient plasmid standard; Figure 5e It is a three-dimensional stereogram of the HIV-1Ψ segment-deficient plasmid standard product of Example 2 in the fluorescence signal channels of GREEN channel (green) + INFRA-RED channel (purple red) + TEAL channel (light blue); Figure 5f It is a three-dimensional stereogram of the HIV-1Ψ segment-deficient plasmid standard product of Example 2 in the BLUE channel (blue) + INFRA-RED channel (purple red) + TEAL channel (light blue) fluorescence signal channels;

[0064] Figure 6aThis is a partial droplet display diagram of the BLUE channel (blue) when detecting HIV-1env segment-deficient plasmid standard in Example 2; Figure 6b This is a partial droplet display diagram of the GREEN channel (green) of Example 2 when detecting HIV-1env segment-deficient plasmid standard; Figure 6c This is a partial droplet display diagram of the INFRA-RED channel (purple) of Example 2 when detecting HIV-1env segment-deficient plasmid standards; Figure 6d This is a partial droplet display diagram of the TEAL channel (light blue) of Example 2 when detecting HIV-1env segment-deficient plasmid standards; Figure 6e It is a three-dimensional stereogram of the HIV-1env segment-deficient plasmid standard product of Example 2 in the BLUE channel (blue) + GREEN channel (green) + INFRA-RED channel (purple) fluorescence signal channels; Figure 6f It is a three-dimensional stereogram of the HIV-1env segment-deficient plasmid standard product of Example 2 in the BLUE channel (blue) + GREEN channel (green) + TEAL channel (light blue) fluorescence signal channels;

[0065] Figure 7a This is a partial droplet display diagram of the BLUE channel (blue) when detecting HIV-1pol-deficient plasmid standard in Example 2; Figure 7b This is a partial droplet display diagram of the GREEN channel (green) of Example 2 when detecting HIV-1 pol segment-deficient plasmid standard; Figure 7c This is a partial droplet display diagram of the INFRA-RED channel (purple) of Example 2 when detecting HIV-1 pol segment-deficient plasmid standard; Figure 7d This is a partial droplet display diagram of the TEAL channel (light blue) of Example 2 when detecting HIV-1 pol segment-deficient plasmid standard; Figure 7e It is a three-dimensional stereogram of the HIV-1 pol segment-deficient plasmid standard product of Example 2 in the BLUE channel (blue) + GREEN channel (green) + TEAL channel (light blue) fluorescence signal channels; Figure 7f It is a three-dimensional stereogram of the HIV-1 pol segment-deficient plasmid standard product of Example 2 in the BLUE channel (blue) + INFRA-RED channel (purple red) + TEAL channel (light blue) fluorescence signal channels;

[0066] Figure 8 This is a schematic diagram of the fluorescent channels selected when scanning the complete RPP30 gene in Example 3;

[0067] Fig. 9 This is a schematic diagram of the fluorescent channel selection when scanning the 5' end segment after the RPP30 gene is broken in Example 3;

[0068] Fig.10 This is a schematic diagram of the fluorescent channel selection when scanning the 3' end segment after the RPP30 gene is broken in Example 3; DETAILED DESCRIPTION

[0069] In order to facilitate the understanding of the present application, in order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present application, and the preferred implementation methods of the present application are given in the accompanying drawings. However, the present application can be implemented in many different forms and is not limited to the implementation methods described herein. On the contrary, the purpose of providing these implementation methods is to make the disclosure of the present application more thoroughly understood. The present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited to the specific embodiments disclosed below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art belonging to the technical field of the present application. The terms used herein are only for the purpose of describing specific implementation methods and are not intended to limit the present application.

[0070] This application is explained by taking the detection of HIV-1 nucleic acid integrity as an example. Of course, it should be understood that the design concept of primers and probes in this application is not limited to the detection of HIV-1 nucleic acid integrity, but can also be used to detect the integrity of nucleic acids of other species.

[0071] Specifically, a plurality of primers and probes containing degenerate bases were designed for the easily breakable segment of HIV-1 nucleic acid, including forward primers Ψ-f, pol-5'f, pol-3'f, env-1f, env-2f, RPP30-1f, RPP30-2f; reverse primers Ψ-r, pol-5'r, pol-3'1r, pol-3'2r, env-r, RPP30-1r, RPP30-2r; probes Ψ-p, pol-5'p, pol-3'p, env-1p, env-2p, env-3p, RPP30-1p, RPP30-2p;

[0072] The sequence of Ψ-f is: 5′-CAGGACTCGGCTTGCTGARG-3′;

[0073] The sequence of pol-5′f is: 5′-GYCCYATTGADACTGTAC-3′;

[0074] The sequence of pol-3′f is: 5′-ARGGAYTATGGAAAACA-3′;

[0075] The sequence of env-1f is: 5′-AGCAGTGGGAMTAGGA-3′;

[0076] The sequence of env-2f is: 5′-ACTATGGGCGCRGCGTC-3′;

[0077] The sequence of RPP30-1f is: 5′-CATGCTCTGTGGTATAGC-3′;

[0078] The sequence of RPP30-2f is: 5′-AGATTTGGACCTGCGAGCG-3′;

[0079] The sequence of Ψ-r is: 5′-TCTCCTTCTAGCCTCC-3′;

[0080] The sequence of pol-5′r is: 5′-ATGGCCAYTGTTTAACYT-3′;

[0081] The sequence of pol-3′1r is: 5′-GTKCCATGTTYTAATCC-3′;

[0082] The sequence of pol-3′2r is: 5′-ATTCCATGTTCTAATCT-3′;

[0083] The sequence of env-r is: 5′-TGCTGYTGCACTATRC-3′;

[0084] The sequence of RPP30-1r is: 5′-GCTGATTTTGAAGAATTTCTG-3′;

[0085] The sequence of RPP30-2r is: 5′-GAGCGGCTGTCTCCACAAGT-3′;

[0086] The sequence of Ψ-p is: 5′-TTTTGGCGTACTCACCAGT-3′;

[0087] The sequence of pol-5′p is: 5′-AAGCCAGGAATGGATGGMC-3′;

[0088] The pol-3′p sequence is: 5′-ATCCTGTCTACYTGCCACAC-3′;

[0089] The sequence of env-1p is: 5′-CCTTGGGTTCTTGGG-3′;

[0090] The sequence of env-2p is: 5′-CCTTAGGTTCTTAGGAGC-3′;

[0091] The sequence of env-3p is: 5′-CTGGCCTGTACCGTCAG-3′;

[0092] The sequence of RPP30-1p is: 5′-TGCCAGCATGATTGTCTCAGC-3′;

[0093] The sequence of RPP30-2p is: 5′-TTCTGACCTGAAGGCTCTGCGCG-3′;

[0094] It should be noted that the degenerate base M shown in the above sequence replaces nucleotide A or C, the degenerate base R replaces nucleotide A or G, the degenerate base Y replaces nucleotide C or T, the degenerate base K replaces nucleotide G or T, and the degenerate base D replaces nucleotide G or A or T.

[0095] Furthermore, Ψ-p is fluorescently labeled with 5'-FAM and 3'-MGB; pol-5'p is fluorescently labeled with 5'-TAMRA and 3'-BHQ2; pol-3'p is fluorescently labeled with 5'-Cy5.5 and 3'-BHQ3; env-1p is fluorescently labeled with 5'-VIC and 3'-MGB; env-2p is fluorescently labeled with 5'-Unlabeled and 3'-MGB; env-3p is fluorescently labeled with 5'-VIC and 3'-MGB; RPP30-1p is fluorescently labeled with 5'-ROX and 3'-BHQ2; and RPP30-2p is fluorescently labeled with 5'-Cy5 and 3'-BHQ3.

[0096] In this embodiment, different fluorescent dyes are used to label different probes. When probes labeled with different fluorescent dyes are excited in droplets, the fluorescent signals generated are different in color. Therefore, positive droplets containing fluorescent signals of four, three, two, one, etc. different color combinations are shown on the three-dimensional stereogram of the fluorescent signal. During data analysis, complete HIV-1 nucleic acid and different types and quantities of defective HIV-1 nucleic acid can be distinguished based on the number of positive droplets with fluorescent signals of different color combinations.

[0097] The negative control substance in this example is nuclease-free water, and the positive control substance is a fragmented plasmid standard substance, and the fragmented plasmid standard substances include the following two types: HIV-1-FL plasmid and RPP30-FL plasmid.

[0098] Specifically, the sample of the kit is selected from whole blood or peripheral blood mononuclear cell (PBMC) samples.

[0099] Specifically, the kit further includes a digital PCR amplification enzyme premix and a fluorescein sodium salt solution, wherein the digital PCR amplification enzyme premix includes dNTPs, Taq enzyme and digital PCR buffer.

[0100] In a specific application, the forward primer, reverse primer and probe are first diluted to 100 μM respectively. Then, all forward primers and reverse primers are prepared into HIV-1 primer-probe mixed solution at a final concentration of 5 μM, and all probes are prepared into a final concentration of 2.5 μM, and then the digital PCR reaction system is prepared. The preparation of the reaction system is shown in Table 3.

[0101] Table 3. Digital PCR reaction system

[0102] Components Single reaction well (μL) Digital PCR Amplification Enzyme Master Mix 5 Fluorescein sodium salt solution 1 HIV-1 Primer Probe Mix 2 sample 10 <![CDATA[ddH2O]]> 7 Total volume 25

[0103] Specifically, the digital PCR reaction program is shown in Table 4.

[0104] Table 4. Digital PCR reaction program

[0105]

[0106] Specific:

[0107] Add 25 μL of digital PCR reaction system to one reaction well of the digital PCR chip;

[0108] The digital PCR chip is placed in a digital PCR instrument to perform PCR amplification to obtain an amplification result;

[0109] The digital PCR chip that obtains the amplification result is placed in a chip scanner for fluorescence signal scanning. Each type of fluorescence signal has a corresponding fluorescence value. A specific fluorescence threshold and fluorescence compensation parameter are substituted into the fluorescence value to obtain the number of positive droplets in each fluorescence channel and the concentration of each target copy number, and to interpret the results of nucleic acid integrity.

[0110] Specific experimental process:

[0111] Primer and probe dilution: First, dilute the forward primer, reverse primer and probe to 100 μM respectively. Then prepare HIV-1 primer-probe mixture with a final concentration of 5 μM for all forward primers and reverse primers and a final concentration of 2.5 μM for all probes, and then prepare the digital PCR reaction system. The preparation of the reaction system is shown in Table 5.

[0112] Table 5. Digital PCR reaction system

[0113] Components 1 reaction well (μL) Digital PCR Amplification Enzyme Master Mix 5 Fluorescein sodium salt solution 1 HIV-1 Primer Probe Mix 2 sample 10 <![CDATA[ddH2O]]> 7 Total volume 25

[0114] Plasmid dilution: First, prepare the plasmid powder into a 10 ng / μL plasmid solution, then use DNA diluent to perform 10-fold gradient dilution, a total of 6 dilution gradients, and set the value and select the use concentration.

[0115] Digital PCR was performed using the Naica six-channel droplet chip digital PCR instrument (Stilla).

[0116] The digital PCR reaction program is shown in Table 6.

[0117] Table 6. Digital PCR reaction program

[0118]

[0119] In a specific experimental example, quadruple positive droplets of intact HIV-1 nucleic acid, triple positive droplets, double positive droplets, single positive droplets of defective HIV-1 nucleic acid, double positive droplets of intact RPP30 internal reference gene, single positive droplets of 5' end segment of RPP30 gene, and single positive droplets of 3' end segment of RPP30 gene, the results are as follows: Figure 1 and as shown in Table 7. Figure 1 It can be seen that the PCR amplification reagents, kits and methods for detecting the integrity of HIV-1 nucleic acid of the present application can better distinguish between complete and defective HIV-1 nucleic acids; and the fluorescence signals detected by the PCR all have corresponding fluorescence values. Specific fluorescence thresholds and fluorescence compensation parameters are substituted into the fluorescence values ​​to obtain the number of positive droplets and copy number concentrations of each target nucleic acid, calculate the shear coefficient, and further calculate more detailed data such as the number of nucleated cells and the intact HIV-1 nucleic acid load through the shear coefficient.

[0120] Table 7. Positive droplets containing intact HIV-1 nucleic acid, defective HIV-1 nucleic acid and RPP30 internal reference gene with different fluorescent signal colors and analysis methods

[0121]

[0122]

[0123]

[0124]

[0125] In the present application, the detection method is applied to the detection of HIV-1 nucleic acid integrity, which has the following advantages: First, the present application adopts six-channel digital PCR, and PCR amplification is simultaneously performed on four segments of HIV-1 nucleic acid and two segments of internal reference genes in one digital PCR reaction well. The detection probes of the four segments of nucleic acid and the two segments of internal reference genes are labeled with different fluorescent dyes, and the amplification of multiple segments of nucleic acid will not produce cross-reactions. After that, by dividing and analyzing the droplets with different fluorescent signals after amplification, not only the number of intact HIV-1 nucleic acids can be accurately determined, but also the HIV-1 nucleic acid packaging signal ψ segment, pol 5' end segment, pol The number of various defective HIV-1 nucleic acids such as defects in the 3' end segment and env segment and hypermutations overcomes the shortcomings of the prior art such as few defective types detected, long detection time, large sample usage, large number of detection holes used, and high cost. It not only improves the detection efficiency and accuracy, but also reduces the detection cost and sample usage; secondly, the present application adds a detection method and calculation method for the internal reference gene for calculating the shear coefficient, and further calculates the number of nucleated cells, the intact HIV-1 nucleic acid load and other more detailed data for user reference by calculating the shear coefficient.

[0126] The present application is described below in conjunction with specific embodiments.

[0127] Example 1: Establishment of a complete HIV-1 nucleic acid detection method

[0128] The purpose of this example is to establish a method for detecting intact HIV-1 nucleic acid by testing a standard substance.

[0129] 1. Primers and probes used in this example: In the early stage of this application, primers and probes for detecting HIV-1 nucleic acid ψ segment, pol 5' end segment, pol 3' end segment and env segment were designed by comparing 742 HIV-1 full-length genome sequences published on the Chinese HIV gene sequence data platform and analyzing the segments where HIV-1 nucleic acid is prone to breakage (such as Figure 2 The segment indicated by the black arrow) and the primer set and probe set for detecting the nucleic acid integrity of human immunodeficiency virus HIV-1 were screened out through a large number of experimental studies, and the nucleotide sequences are shown in Table 8.

[0130] Table 8. Primer and probe sequence list for HIV-1 nucleic acid integrity detection

[0131]

[0132] As shown in Table 8, the degenerate base M replaces A or C, the degenerate base R replaces A or G, the degenerate base Y replaces C or T, the degenerate base K replaces G or T, and the degenerate base D replaces G or A or T.

[0133] The forward primer, reverse primer and probe used to amplify the HIV-1 nucleic acid ψ segment, pol 5' end segment, pol 3' end segment and env segment were first diluted to 100 μM respectively. Then, all forward primers and reverse primers were prepared at a final concentration of 5 μM, and all probes were prepared at a final concentration of 2.5 μM to form HIV-1 primer-probe mixed solution-1, and then the digital PCR reaction system was prepared.

[0134] 2. The standard used in this example is HIV-1-FL plasmid, which is a plasmid containing the complete HIV-1 nucleic acid sequence (HIV-1 nucleic acid sequence HXB2 (K03455): 1-9719) (e.g. Figure 2 In this example, HIV-1-FL plasmid powder was first prepared into a 10 ng / μL plasmid solution, and then a DNA diluent was used to perform a 10-fold gradient dilution, a total of 6 gradients were diluted, and digital PCR was used to determine the value of HIV-1-FL plasmid. According to the result of the determination, a concentration of about 1000 cp / μL was selected as a high-concentration HIV-1-FL plasmid template and 100 cp / μL was selected as a low-concentration HIV-1-FL plasmid template for digital PCR detection, and the detection was repeated three times.

[0135] 3. Prepare the complete HIV-1 nucleic acid detection digital PCR reaction solution according to Table 9

[0136] Table 9. Preparation of complete HIV-1 nucleic acid digital PCR reaction solution

[0137]

[0138]

[0139] 4. Add 25 μL of digital PCR reaction solution to the reaction wells of the chip.

[0140] 5. Transfer the chip to Naica TM In the Geode droplet generation and amplification system, droplet generation and PCR amplification were performed according to the digital PCR reaction program in Table 10.

[0141] Table 10. Digital PCR reaction program

[0142]

[0143] 6. Data reading: After the digital PCR reaction is completed, place the chip in Naica TM In the prism6 droplet reading and analysis system, fluorescence signals are collected and the results are automatically saved.

[0144] 7. Obtain the test results: Use Crystal Miner software to open the data, adjust the threshold line according to the fluorescence values ​​of the six channels: BLUE, TEAL, GREEN, INFRA-RED, RED and YELLOW, and then follow the steps in the “Positivity Combination” interface of Crystal Miner software. Figure 3 As shown, the BLUE, TEAL, GREEN, and INFRA-RED fluorescence channels are selected as positive, and the YELLOW and RED fluorescence channels are selected as unrestricted, and then the number of droplets and the copy number concentration with positive BLUE, TEAL, GREEN, and INFRA-RED fluorescence signals can be obtained, which represents the number of positive droplets and the copy number concentration of the plasmid containing the complete HIV-1 nucleic acid. The software will automatically display the number of positive droplets and the copy number concentration of each channel, representing the number of positive droplets and the copy number concentration of the ψ segment, pol 5' end segment, pol 3' end segment, and env segment of the HIV-1 nucleic acid, respectively (as shown in Tables 11 and 12).

[0145] Table 11. Digital PCR test results of complete HIV-1 plasmid standard

[0146]

[0147]

[0148] Table 12. Summary of test results of complete HIV-1 plasmid standards

[0149]

[0150] 8. Analysis of complete HIV-1 nucleic acid test results: Figure 4a-4g As shown, Figure 4a The figure shows the status of all droplets after a reaction well is scanned after digital PCR amplification is completed. Figure 4b-4e for Figure 4a The enlarged image of the white framed area. In the same white framed area, in the BLUE channel (blue) ( Figure 4b )、GREEN channel(green)( Figure 4c )、INFRA-RED channel (purple) ( Figure 4d )、TEAL channel (light blue) ( Figure 4e ) is a distribution diagram of negative droplets and positive droplets, wherein the white bright spots in the figure are positive droplets, representing that the droplets contain the amplified fragments of the target, and the dark spots are negative droplets, representing that the droplets do not contain the amplified fragments of the target. Figure 4b-4eIt is shown that the positions of the positive droplets of the reaction wells containing HIV-1-FL plasmid after PCR amplification is completed and scanned under four fluorescence channels (BLUE, GREEN, INFRA-RED, TEAL) completely overlap, indicating that when the HIV-1-FL plasmid is distributed to the same droplet, after PCR amplification, the fluorescent probes that simultaneously bind to the four segments of ψ, pol 5' end, pol 3' end, and env on the HIV-1-FL plasmid are hydrolyzed and release corresponding fluorescent signals. Therefore, four fluorescent signals will be displayed simultaneously in the same droplet, namely BLUE channel (blue) + GREEN channel (green) + INFRA-RED channel (purple red) + TEAL channel (light blue), and the positive droplet numbers and copy number concentrations of the complete HIV-1 (HIV-1-FL plasmid), HIV-1ψ segment, HIV-1pol 5' end segment, HIV-1pol 3' end segment, and HIV-1env segment can be obtained (as shown in Tables 11 and 12). When the fluorescence signal of intact HIV-1 nucleic acid is displayed using a three-dimensional stereogram, since four fluorescence signals exist simultaneously in a single droplet, it is composed of two three-dimensional stereograms. Figure 4f and Figure 4g The X-axis, Y-axis, and Z-axis on the three-dimensional graph represent different fluorescence channels. The positive droplets with three-channel fluorescence signals of BLUE (blue) + GREEN (green) + TEAL (light blue) will be in a three-dimensional space with fluorescence values ​​on the X-axis, Y-axis, and Z-axis at the same time ( Figure 4f black box), and Figure 4gThe positive droplets shown in the black box that simultaneously have INFRA-RED (purple red) fluorescence signals in BLUE (blue) + INFRA-RED (purple red) + TEAL (light blue) constitute four positive droplets of BLUE channel (blue) + GREEN ventilation (green) + INFRA-RED channel (purple red) + TEAL channel (light blue). Then, the copy number of the complete HIV-1 nucleic acid is calculated according to the number of the four positive droplets, that is, as shown in Tables 11 and 12, it can be seen that the copy number concentration of the complete HIV-1 nucleic acid of the high-concentration HIV-1-FL plasmid is 0.94-0.99 to the copy number concentration of the ψ segment, pol 5' end segment, pol 3' end segment, and env segment of HIV-1. The ratio is about 1, and the relative deviation (B) is less than 10%, indicating that the amplification specificity for the high-concentration HIV-1-FL plasmid is good, and no cross-amplification reaction between different nucleic acid segments occurs in the sample at high concentration. The copy number concentration ratio of the complete HIV-1 nucleic acid in the low-concentration HIV-1-FL plasmid to the copy number concentration of the HIV-1 ψ segment, pol 5' end segment, pol 3' end segment and env segment is 0.84-0.98, which is about 1, and the relative deviation (B) is less than 15%, indicating that the amplification specificity for the low-concentration HIV-1-FL plasmid is also good, and no cross-amplification reaction between different nucleic acid segments occurs in the sample at a low concentration. It also shows that the primers and probes designed for different segments of HIV-1 nucleic acid have similar amplification efficiencies for the corresponding segments in the sample. The detection results of Example 1 also show that this method can accurately identify the complete HIV-1 nucleic acid.

[0151] Example 2: Establishment of a method for detecting defective HIV-1 nucleic acid

[0152] The purpose of this example is to establish a detection method for different defective HIV-1 nucleic acids by testing defective standards.

[0153] 1. The types and preparation methods of primers and probes used in this example are the same as those in Example 1;

[0154] 2. The standards used in this example are HIV-1ΨD plasmid (HIV-1Ψ segment is deleted, containing only HIV-1 nucleic acid sequence HXB (K03455): 2085-9719), HIV-1envD plasmid (HIV-1env segment is deleted, containing only HIV-1 nucleic acid sequence HXB (K03455): 1-5280), HIV-1polD plasmid (HIV-1pol segment is deleted, containing only HIV-1 nucleic acid sequence HXB2 (K03455): 1-2085, 5280-9719), as shown by the arrows in Figure 5. In this example, HIV-1ΨD plasmid, HIV-1envD plasmid, and HIV-1polD plasmid dry powder were first prepared into a 10 ng / μL plasmid solution, and then the three plasmids were gradiently diluted 10 times using a DNA diluent, a total of 6 gradients were diluted, and the three plasmids were quantified using digital PCR. According to the result of the quantification, a plasmid standard with a concentration of about 100 cp / μL was selected as a template for digital PCR detection, and the detection was repeated three times;

[0155] 3. The operation method of this embodiment is the same as that of embodiment 1, except that the templates in the PCR reaction system are replaced with HIV-1ΨD plasmid standard, HIV-1envD plasmid standard, and HIV-1polD plasmid standard respectively;

[0156] 4. Test results of defective HIV-1 nucleic acid plasmid standard:

[0157] 4.1 The results of PCR amplification of HIV-1ΨD plasmid standard are shown in 5a-5f. Figure 5a-5d Displayed in the same field of view, BLUE channel (blue) ( Figure 5a )、GREEN channel(green)( Figure 5b )、INFRA-RED channel (purple) (5c), TEAL channel (light blue) ( Figure 5d ) distribution of negative and positive droplets. In the figure, the white bright spots are positive droplets, indicating that the droplets contain the target amplified fragment, and the dark spots are negative droplets, indicating that the droplets do not contain the target amplified fragment. The positive droplets after PCR amplification and scanning in the reaction well containing the HIV-1ΨD plasmid standard are in the GREEN channel (green) ( Figure 5b )、INFRA-RED channel (purple) ( Figure 5c )、TEAL channel (light blue) ( Figure 5d) The positions under the three fluorescence channels will completely overlap, indicating that when the HIV-1ΨD plasmid is distributed to the same droplet after PCR amplification, due to the loss of the HIV-1Ψ segment, the probe that can bind to the HIV-1Ψ segment (FAM labeled, BLUE channel, blue) cannot recognize the Ψ segment, and only the fluorescent probes that simultaneously bind to the pol 5' end, pol 3' end, and env segments on the HIV-1ΨD plasmid are hydrolyzed and release corresponding fluorescent signals. Therefore, three fluorescent signals will be displayed simultaneously in the same droplet, namely GREEN channel (green) + INFRA-RED channel (purple red) + TEAL channel (light blue), and the positive droplet number and copy number concentration of the HIV-1pol 5' end segment, HIV-1pol 3' end segment, and HIV-1env segment can be obtained (as shown in Table 13). When the fluorescence signal of HIV-1Ψ segment deletion plasmid standard is displayed using a three-dimensional stereogram, since three fluorescence signals exist simultaneously in a single droplet, the X-axis, Y-axis, and Z-axis on the three-dimensional stereogram represent different fluorescence channels respectively. Figure 5e The droplet showing three fluorescent signals of GREEN (green) + INFRA-RED (purple red) + TEAL (light blue) will be in a three-dimensional space with fluorescence values ​​on the X-axis, Y-axis, and Z-axis at the same time ( Figure 5e In the 3D image with BLUE channel ( Figure 5f ), there is no droplet in three-dimensional space due to the lack of fluorescent signal in the BLUE channel (blue).

[0158] 4.2. The results of the PCR amplification of HIV-1envD plasmid standard are as follows Figure 6a-6f As shown, Figure 6a-6d Displayed in the same field of view, BLUE channel (blue) ( Figure 6a )、GREEN channel(green)( Figure 6b )、INFRA-RED channel (purple) ( Figure 6c )、TEAL channel (light blue) ( Figure 6d ) distribution diagram of negative and positive droplets. In the figure, the white bright spots are positive droplets, indicating that the droplets contain the target amplified fragment, and the dark spots are negative droplets, indicating that the droplets do not contain the target amplified fragment. In the reaction well containing HIV-1envD plasmid, the positive droplets after PCR amplification and scanning are in the BLUE channel (blue) ( Figure 6a )、GREEN channel(green)( Figure 6b )、INFRA-RED channel (purple) ( Figure 6c) The positions under the three fluorescence channels will completely overlap, indicating that when the HIV-1envD plasmid is distributed to the same droplet after PCR amplification, due to the loss of the HIV-1env segment, the probe that can bind to the HIV-1env segment (VIC marker, TEAL channel, light blue) cannot recognize the env segment, and only the fluorescent probe that simultaneously binds to the three segments of Ψ, pol5' end, and pol 3' end on the HIV-1envD plasmid is hydrolyzed and releases the corresponding fluorescent signal. Therefore, three fluorescent signals will be displayed simultaneously in the same droplet, namely BLUE channel (blue) + GREEN channel (green) + INFRA-RED channel (purple red), and the number of positive droplets and the copy number concentration of the HIV-1Ψ segment, HIV-1pol 5' end segment, and HIV-1pol 3' end segment can be obtained (as shown in Table 13). When the fluorescence signal of HIV-1env segment deletion plasmid standard is displayed using a three-dimensional stereogram, since three fluorescence signals exist simultaneously in a single droplet, the X-axis, Y-axis, and Z-axis on the three-dimensional stereogram represent different fluorescence channels. Figure 6e The droplet with three fluorescent signals of BLUE channel (blue) + GREEN channel (green) + INFRA-RED channel (purple) will be in a three-dimensional space with fluorescence values ​​on the X-axis, Y-axis and Z-axis at the same time ( Figure 6e black box), while in the 3D image with TEAL channel ( Figure 6f ), there is no droplet in three-dimensional space due to the lack of fluorescent signal in the TEAL channel (light blue).

[0159] 4.3 The results of the PCR amplification of HIV-1polD plasmid standard are as follows Figures 7a-7f As shown, Figures 7a-7d Displayed in the same field of view, BLUE channel (blue) ( Figure 7a )、GREEN channel(green)( Figure 7b )、INFRA-RED channel (purple) ( Figure 7c )、TEAL channel (light blue) ( Figure 7d ) distribution of negative and positive droplets. In the figure, the white bright spots are positive droplets, indicating that the droplets contain the target amplified fragment, and the dark spots are negative droplets, indicating that the droplets do not contain the target amplified fragment. The positive droplets in the reaction well containing HIV-1polD plasmid after PCR amplification are scanned in the BLUE channel (blue) ( Figure 7a )、TEAL channel (light blue) ( Figure 7d) The positions under the two fluorescence channels will completely overlap, indicating that when the HIV-1polD plasmid is distributed to the same droplet and amplified by PCR, due to the loss of the HIV-1pol segment, the probes that can bind to the HIV-1pol segment (labeled with TAMRA and Cy5.5, GREEN channel and INFRA-RED channel, green and purple) cannot recognize the pol segment. Only the fluorescent probes that bind to the Ψ and env segments on the HIV-1polD plasmid are hydrolyzed and release the corresponding fluorescent signals. Therefore, two fluorescent signals will be displayed simultaneously in the same droplet, namely the BLUE channel (blue) + TEAL channel (light blue), and the number of positive droplets and the copy number concentration of the HIV-1Ψ segment and HIV-1env segment can be obtained (as shown in Table 13). When a three-dimensional stereogram is used to display the fluorescent signal of the HIV-1pol segment-deficient plasmid standard, due to the simultaneous presence of two fluorescent signals in a single droplet, the three-dimensional stereogram Figure 7e The X-axis, Y-axis, and Z-axis represent the fluorescence channels of BLUE, TEAL, and GREEN, respectively. The positive droplets with only two fluorescence signals of BLUE channel (blue) + TEAL channel (light blue) ( Figure 7e Black box); three-dimensional Figure 7f The X-axis, Y-axis, and Z-axis represent the fluorescence channels of BLUE, TEAL, and INFRA-RED, respectively. The positive droplets only have two fluorescence signals: BLUE channel (blue) + TEAL channel (light blue). Figure 7f Table 13 shows the copy number concentration of defective HIV-1 nucleic acid plasmid standards.

[0160] Table 13. Defective HIV-1 nucleic acid plasmid standard copy number concentration results

[0161]

[0162]

[0163] As shown in Table 13, in the plasmid lacking the ψ segment of HIV-1 nucleic acid, the copy number concentrations of the pol 5' segment, pol 3' segment, and env segment were detected, but the copy number concentration of the ψ segment was not detected. In the plasmid lacking the env segment of HIV-1 nucleic acid, only the copy number concentrations of the ψ segment, pol 5' segment, and pol 3' segment were detected, but the copy number concentration of the env segment was not detected. In the plasmid lacking the pol 5' segment and pol 3' segment of HIV-1 nucleic acid, only the copy number concentrations of the ψ segment and env segment were detected, but the copy number concentrations of the pol 5' segment and pol 3' segment were not detected. The above results indicate that this method can accurately distinguish HIV-1 nucleic acids of different defective types, such as nucleic acids lacking the HIV-1 ψ, pol, and env segments.

[0164] Example 3: Obtaining and verifying the shear coefficient

[0165] The purpose of this example is to obtain the shear coefficient of nucleic acid by detecting the internal reference RPP30 gene to correct the problem of deviation in nucleic acid integrity detection caused by nucleic acid breakage during the extraction process.

[0166] 1. The primers and probes used in this example are designed for the 5' and 3' end segments of the RPP30 gene, and their nucleotide sequences are shown in Table 14.

[0167] Table 14. Primer and probe sequences of RPP30 gene

[0168]

[0169]

[0170] In this example, the forward primer, reverse primer and probe used to amplify the 5' and 3' end segments of the RPP30 gene were first diluted to 100 μM, respectively, and then all forward primers and reverse primers were prepared into an RPP30 primer-probe mixture at a final concentration of 5 μM and all probes were prepared at a final concentration of 2.5 μM to prepare the digital PCR reaction system.

[0171] 2. The standard products used in this example are RPP30-FL plasmid (a plasmid containing the complete RPP30 gene sequence, containing the sequence GRCh37 / hg19 chr10:92631758-92639394), RPP30-5F plasmid (a plasmid containing the 5' end sequence of the RPP30 gene), and RPP30-3F plasmid (a plasmid containing the 3' end sequence of the RPP30 gene). In this example, the plasmid dry powder was first prepared into a 10 ng / μL plasmid solution, and then a DNA diluent was used for 10-fold gradient dilution, a total of 6 gradients were diluted, and the three plasmids were quantified using digital PCR. According to the quantification results, three plasmids with a concentration of about 3000 cp / μL of each plasmid were selected and mixed in a ratio of 1:1:1 as a standard product simulating a breakage ratio of 50% and repeated three times.

[0172] 3. The preparation and reaction procedure of the digital PCR reaction system are shown in Tables 15 and 16;

[0173] Table 15. Preparation table of RPP30 gene digital PCR reaction system

[0174] Components Single reaction well (μL) Digital PCR Amplification Enzyme Master Mix 5 Fluorescein sodium salt solution (2.5 μM) 1 RPP30 Primer Probe Mix 2 1:1:1 mixture of RPP30-FL plasmid, RPP30-5F plasmid, and RPP30-3F plasmid 2 <![CDATA[ddH2O]]> 15 Total volume 25

[0175] Table 16. Digital PCR reaction program

[0176]

[0177]

[0178] 4. Data reading: After the digital PCR reaction is completed, place the chip in Naica TM In the prism6 droplet reading and analysis system, fluorescence signals are collected and the results are automatically saved.

[0179] 5. Obtaining the shear coefficient: Open the Crystal Miner software and set it in the "PositivityCombination" interface of the software, such as Figure 8 The operation shown in the figure selects the YELLOW (yellow) and RED (red) fluorescence channels as positive, and selects other fluorescence channels as unrestricted (i.e., either positive or negative), and obtains the number of droplets and copy number concentrations with both YELLOW (yellow) and RED (red) fluorescence channels positive. This result is the number of positive droplets and copy number concentrations containing the complete RPP30 gene in the test sample. Fig. 9The operation shown in the figure selects the RED (red) fluorescence channel as positive, the YELLOW (yellow) fluorescence channel as negative, and other fluorescence channels as unrestricted (i.e., either positive or negative), and the number and copy number concentration of droplets with the RED (red) fluorescence channel as positive and the YELLOW (yellow) fluorescence channel as negative can be obtained. This result is the number and copy number concentration of positive droplets containing the 5' end amplified fragment after the RPP30 gene is broken in the test sample. Fig.10 The operation shown in the figure, select the YELLOW (yellow) fluorescence channel as positive, the RED (red) fluorescence channel as negative, and select other fluorescence channels as unrestricted (i.e., either positive or negative), and the number and copy number concentration of droplets with both positive YELLOW (yellow) fluorescence channel and negative RED (red) fluorescence channel can be obtained. This result is the number and copy number concentration of positive droplets containing the 3' end amplified fragment after the RPP30 gene is broken in the test sample. As shown in Table 17, the results are the number and copy number concentration of positive droplets of the intact RPP30 gene, the 5' end segment after the break, and the 3' end segment after the break. The calculation results of the shear coefficient are shown in Table 18.

[0180] Table 17. Detection results of the shear coefficient standard RPP30 gene

[0181]

[0182]

[0183] 6. Calculation formula of shear coefficient:

[0184]

[0185] Table 18. Calculation results of shear coefficient

[0186]

[0187] Example 4: Application of shear coefficient in standard products

[0188] The purpose of this example is to determine the application effect of the shear coefficient by testing HIV-1-FL plasmid standard.

[0189] 1. The primer and probe sequences used in this example are shown in Tables 8 and 14. All forward primers, reverse primers and probes shown in SEQ ID NO.1 to SEQ ID NO.22 were first diluted to 100 μM, and then all forward primers and reverse primers were prepared at a final concentration of 5 μM, and all probes were prepared at a final concentration of 2.5 μM to prepare an HIV-1 primer-probe mixture for the preparation of a digital PCR reaction system.

[0190] 2. The standard samples used in this example are HIV-FL plasmid and RPP30-FL plasmid, which are fully mixed in a certain ratio and then broken by ultrasonic treatment to simulate the nucleic acid breakage phenomenon during nucleic acid extraction. The test is repeated 5 times to verify the effect of the shear coefficient.

[0191] 3. The preparation and reaction procedure of the digital PCR reaction system are shown in Tables 19 and 20;

[0192] Table 19. Preparation table of digital PCR reaction system for shearing system verification

[0193] Components Single reaction well (μL) Digital PCR Amplification Enzyme Master Mix 5 Fluorescein sodium salt solution (2.5 μM) 1 HIV-1 Primer Probe Mix 2 HIV-FL plasmid and RPP30-FL plasmid were mixed and sonicated 2 <![CDATA[ddH2O]]> 15 Total volume 25

[0194] Table 20. Digital PCR reaction program

[0195]

[0196]

[0197] 4. Read the results: After the digital PCR reaction is completed, place the chip in the Naica TM In the prism6 droplet reading and analysis system, choose to open the HIV-1 fluorescence signal acquisition file, which has been pre-stored in the scanning software. It contains the "settings for the number of positive droplets and copy number concentrations of intact HIV-1 nucleic acid" in Example 1 and the "settings for obtaining the parameters required for the shear coefficient" in Example 3. Collect the fluorescence signal and automatically save the scanning results.

[0198] 5. The test results of this embodiment are shown in Table 21, Table 22 and Table 23.

[0199] Table 21. HIV-1-FL plasmid standard test results

[0200]

[0201] Table 22. Test results of RPP30 plasmid standard

[0202]

[0203]

[0204] The calculation formula of shear coefficient is:

[0205]

[0206]

[0207] Table 23. Conversion table for intact HIV-1 nucleic acid with added shear coefficient

[0208]

[0209] 6. The results showed that when the complete HIV-FL plasmid standard and the complete RPP30-FL plasmid standard were mixed and then ultrasonically treated, the shearing effect of ultrasonic treatment on DNA was random, so the plasmid after ultrasonic treatment would have various segment types similar to HIV-1 nucleic acid. After digital PCR amplification, droplets with various types of fluorescent signals, single positive droplets (RED) containing the 5' end amplified fragment of the RPP30 gene, single positive droplets (YELLOW) containing the 3' end amplified fragment, and double positive droplets (RED+YELLOW) containing the complete RPP30 gene would be displayed on the three-dimensional stereograms of BLUE (X-axis), TEAL (Y-axis), GREEN (Z-axis) and BLUE (X-axis), TEAL (Y-axis), INFRA-RED (Z-axis). The results of the copy numbers of various HIV-1 nucleic acid segments calculated based on the number of positive droplets are shown in Table 21. The concentration of the complete HIV-FL plasmid standard is 14.14 cps / μL, which is lower than the copy number concentrations of HIV-1ψ segment, pol 5' segment, pol 3' segment, and env segment, but the copy number concentrations of ψ segment, pol 5' segment, pol 3' segment, and env segment are similar, all between 23.28 and 25.4 cps / μL. Through the correction of the RPP30 shear coefficient, as shown in Table 23, the true integrity HIV-FL concentration can be obtained as 24.20 (CV value = 8.01%), which is consistent with the concentrations of ψ segment, pol segment, pol 3' segment, and env segment. This result shows that the correction of the shear coefficient will eliminate the low concentration phenomenon caused by nucleic acid breakage during nucleic acid extraction, and has a good application effect.

[0210] Example 5: A kit for detecting the integrity of human immunodeficiency virus (HIV-1) nucleic acid

[0211] 1. The components of a kit for detecting nucleic acid integrity of human immunodeficiency virus (HIV-1) are shown in Table 24.

[0212] Table 24. Composition of Human Immunodeficiency Virus (HIV-1) Nucleic Acid Integrity Detection Kit

[0213] Serial number Component name Specification quantity 1 Digital PCR Amplification Enzyme Master Mix 120μL 1 tube 2 Fluorescein sodium salt solution 50μL 1 tube 3 HIV-1 Primer Probe Mix 48μL 1 tube 4 HIV-1 Negative Control 300μL 1 tube 5 HIV-1 Positive Control 60μL 1 tube

[0214] Among them, the digital PCR amplification enzyme premix contains dNTPs, Taq enzyme and digital PCR buffer;

[0215] The fluorescein sodium salt solution was a 2.5 μM fluorescein sodium salt solution;

[0216] The digital PCR reaction solution is a mixture of primers and probes for amplifying the ψ segment, pol 5' segment, pol 3' segment, env segment, and RPP30 gene 5' segment and 3' segment of HIV-1 nucleic acid, with each primer having a concentration of 5 μM and each probe having a concentration of 2.5 μM;

[0217] HIV-1 negative control was ddH2O;

[0218] The HIV-1 positive control was a fragmented plasmid standard. The fragmented plasmid standard was a mixture of HIV-1-FL plasmid and RPP30-FL plasmid, which were subjected to ultrasonic fragmentation treatment and then identified.

[0219] 2. A method for using a human immunodeficiency virus (HIV-1) nucleic acid integrity detection kit

[0220] 2.1 Instrument: Applicable to Naica six-channel microdroplet chip digital PCR (Stilla).

[0221] 2.2 Sample Type: The kit of the present application is suitable for human whole blood or peripheral blood mononuclear cell (PBMC) samples.

[0222] 2.3.1 Sample processing

[0223] The nucleic acid in the sample was extracted using a commercially available kit. For specific operations, please refer to the product instructions in the commercially available kit. The extracted nucleic acid can be directly used in subsequent experiments.

[0224] 2.3.2 Preparation of digital PCR reaction system: Take out the digital PCR amplification enzyme premix, fluorescein sodium salt solution, digital PCR reaction solution, HIV-1 positive control, and HIV-1 negative control from the kit, thaw at room temperature and shake to mix, centrifuge at 3000 rpm for 10 seconds before use, and prepare the digital PCR reaction system according to Table 25.

[0225] Table 25. Digital PCR reaction system

[0226] Components Single well aliquot (μL) Digital PCR Amplification Enzyme Master Mix 5 Fluorescein sodium salt solution 1 HIV-1 Primer Probe Mix 2 Samples (both negative and positive controls) 10 Total volume <![CDATA[Make up to 25 with ddH2O]]>

[0227] 2.3.3 Sample loading, droplet generation, and digital PCR amplification

[0228] Add 25 μL of the digital PCR reaction solution prepared according to Table 25 to the sample well of the digital PCR chip and cover it with a lid. The digital PCR chip is a Sapphire chip;

[0229] Transfer the chip to Naica TMIn the Geode droplet generation and amplification system, the digital PCR reaction program was set according to Table 26, and droplet generation and PCR amplification were performed.

[0230] Table 26. Digital PCR reaction program

[0231]

[0232] 2.3.4 Reading results: After the digital PCR reaction is completed, place the chip in the Naica TM In the prism6 droplet reading and analysis system, the fluorescence signal is collected according to step 4 in Example 4, and the results can be automatically saved.

[0233] 3. Result analysis and interpretation

[0234] Data analysis: Crystal Miner software was used to open the data, and the threshold line was adjusted according to the fluorescence signal values ​​of the BLUE (blue), TEAL (light blue), GREEN (green), INFRA-RED (purple red), RED (red), and YELLOW (yellow) channels to obtain the positive droplet numbers and copy number concentrations of intact HIV-1 nucleic acid (the copy number concentration corresponding to the four positive droplets of BLUE+GREEN+INFRA-RED+TEAL), intact RPP30, the 5' end segment after RPP30 breakage, and the 3' end segment after RPP30 breakage.

[0235]

[0236]

[0237]

[0238] The calculated intact HIV-1 nucleic acid load per million cells is the abundance of intact HIV-1 nucleic acid in the current HIV-1 latent reservoir.

[0239] Example 5: Detection of clinical samples

[0240] This example further verifies the effect of the human immunodeficiency virus (HIV-1) nucleic acid integrity detection kit of the present application. This example uses 15 clinical nucleic acid samples, including 5 DNA extracted from normal human PBMC and 10 samples containing HIV latent reservoirs detected by VOA.

[0241] 1. The detection kit and usage were performed as described in Example 4. The detection results are shown in Table 27.

[0242] Table 27. Digital PCR test results of HIV-1 nucleic acid integrity of clinical samples

[0243]

[0244]

[0245] As shown in Table 27, the results of the detection of 15 clinical samples using the method and kit for detecting the integrity of human immunodeficiency virus (HIV-1) nucleic acid described in this application in this embodiment are that there are 10 samples with intact HIV-1 nucleic acid and 5 negative samples, which are consistent with the detection results of VOA, with a compliance rate of 100% and an accuracy rate of 100%. At the same time, the calculation formula of the shear coefficient and the intact HIV-1 nucleic acid load in one million cells described in this application was used to obtain the data of the shear coefficient and the intact HIV-1 nucleic acid load in one million cells of 15 samples. It was found that for 15 clinical samples, the measured values ​​of the intact HIV-1 nucleic acid copy number concentration substituted with the shear coefficient were all higher than the intact HIV-1 nucleic acid copy number concentration without adding the shear coefficient. It is explained that the breakage of nucleic acids will lead to a decrease in the concentration of intact nucleic acids, and the state of nucleic acid breakage of different samples is also significantly different, so adding the shear coefficient can better reflect the true copy number concentration of intact HIV-1 nucleic acid in the unextracted sample.

[0246] The results of the above examples show that the method and kit for detecting the integrity of human immunodeficiency virus (HIV-1) nucleic acid described in the present application can be accurately used for detecting the integrity of human immunodeficiency virus (HIV-1) nucleic acid. The present application is simple to operate, has a short detection time (detection is completed within 3 hours), and uses a small amount of sample (only 2 to 10 mL of blood is required to achieve simultaneous detection of four segments of HIV-1 nucleic acid and two segments of RPP30 gene in a single digital PCR reaction well, and the amplification of nucleic acid in each segment will not affect the amplification of other segments. The method and kit can not only accurately detect complete HIV-1 nucleic acid, but also can simply and quickly detect HIV-1 nucleic acid of different defective types (as shown in Example 2).

[0247] The present application has at least the following advantages: First, the present application adopts six-channel digital PCR, which can realize simultaneous PCR amplification of at least four segments of HIV-1 nucleic acid and two segments of RPP30 gene in a single digital PCR reaction well. The test results show that the detection concentration ratio after amplification of multiple segments of nucleic acid is 1:1, indicating that this method will not produce cross-reactions for the simultaneous amplification of multiple segments of nucleic acid. By dividing and analyzing the droplets with different types of fluorescent signals after amplification, not only the number of intact HIV-1 nucleic acids can be accurately determined, but also the number of various defective HIV-1 nucleic acids such as the HIV-1 nucleic acid ψ segment, pol5' end segment, pol 3' end segment, env segment and hypermutation can be determined, overcoming the shortcomings of the prior art such as few defective types detected, long detection time, large sample dosage and high cost, which not only improves the detection efficiency but also reduces the detection cost; secondly, the present application adopts six-channel digital PCR, and while realizing the identification of HIV-1 nucleic acid types in a single digital PCR reaction well, it also adds the detection method and calculation method of the RPP30 gene for calculating the shear coefficient. The shear coefficient is further calculated to further calculate more detailed data such as the number of nucleated cells and the intact HIV-1 nucleic acid load for user reference.

[0248] It should be understood that although the present application specifically designs primers and probes for HIV-1 nucleic acid, those skilled in the art should know that, based on the primers and probes for HIV-1 nucleic acid of the present application, the target pairing sequence can be adjusted accordingly. At the same time, the design concept based on the detection method of the present application is not limited to the corresponding changes given in the present application.

[0249] The technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. It should be noted that "in one embodiment", "for example", "for example", etc. in this application are intended to illustrate the present application, rather than to limit the present application. The above-described embodiments only express several implementation methods of the present application, and the description is relatively specific and detailed, but it cannot be understood as a limitation on the scope of the patent application. It should be pointed out that for a person of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can also be made, which all belong to the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be subject to the attached claims.

Claims

1. A method for detecting nucleic acid integrity based on digital PCR for non-diagnostic purposes, characterized in that: The steps include: A digital PCR reaction system for detecting a sample is prepared using a kit, and the sample is added; wherein the kit includes a plurality of primers and a plurality of probes, and at least some of the primers and probes contain degenerate bases; the 5' ends of the plurality of probes are labeled with a fluorescent dye that is different from the 5' ends of other probes, so that after the plurality of probes are amplified by PCR in the same digital PCR reaction well, the amplification products can be distinguished by positive droplets that generate different types of fluorescent signals; Loading the digital PCR reaction system into the digital PCR chip; The digital PCR chip is placed in a digital PCR instrument for PCR amplification to obtain an amplification result; the digital PCR instrument is a six-channel microdroplet chip digital PCR instrument; The digital PCR chip having the amplification result is placed in a chip scanner to scan the fluorescence signal of the amplified droplets, and the positive droplets containing fluorescent signals of different colors are subjected to fluorescent crosstalk analysis, and then the nucleic acid integrity is judged according to the positive droplets containing different fluorescent signal colors after the analysis; Wherein, the sequences of the plurality of forward primers include Ψ-f, pol-5'f, pol-3'f, env-1f, env-2f, RPP30-1f, RPP30-2f; the sequences of the plurality of reverse primers include Ψ-r, pol-5'r, pol-3'1r, pol-3'2r, env-r, RPP30-1r, RPP30-2r; the plurality of probe sequences include Ψ-p, pol-5'p, pol-3'p, env-1p, env-2p, env-3p, RPP30-1p, RPP30-2p, wherein: The sequence of Ψ-f is: 5′-CAGGACTCGGCTTGCTGARG-3′; The sequence of pol-5'F is: 5′-GYCCYATTGADACTGTAC-3′; The sequence of pol-3'F is: 5′-ARGGAYTATGGAAAACA-3′; The sequence of env-1f is: 5′-AGCAGTGGGAMTAGGA-3′; The sequence of env-2f is: 5′-ACTATGGGCGCRGCGTC-3′; The sequence of RPP30-1f is: 5′-CATGCTCTGTGGTATAGC-3′; The sequence of RPP30-2f is: 5'-AGATTTGGACCTGCGAGCG-3'; the sequence of Ψ-r is: 5′-TCTCCTTCTAGCCTCC-3′; The sequence of pol-5'R is: 5′-ATGGCCAYTGTTTAACYT-3′; The sequence of pol-3'1R is: 5′-GTKCCATGTTYTAATCC-3′; The sequence of pol-3'2R is: 5′-ATTCCATGTTCTAATCT-3′; The sequence of env-r is: 5′-TGCTGYTGCACTATRC-3′; The sequence of RPP30-1r is: 5'-GCTGATTTTGAAGAATTTCTG-3'; the sequence of RPP30-2r is: 5'-GAGCGGCTGTCTCCACAAGT-3'; the sequence of Ψ-p is: 5'-TTTTGGCGTACTCACCAGT-3'; the sequence of pol-5'p is: 5'-AAGCCAGGAATGGATGGMC-3'; pol-3'p sequence is: 5'-ATCCTGTCTACYTGCCACAC-3'; the sequence of env-1p is: 5′-CCTTGGGTTCTTGGGA-3′; The sequence of env-2p is: 5′-CCTTAGGTTCTTAGGAGC-3′; The sequence of env-3p is: 5′-CTGGCCTGTACCGTCAG-3′; The sequence of RPP30-1p is: 5'-TGCCAGCATGATTGTCTCAGC-3'; The sequence of RPP30-2p is: 5'-TTCTGACCTGAAGGCTCTGCGCG-3'; Wherein, the PCR chip is a liquid phase chip; Among them, the fluorescent label of Ψ-p is 5'-FAM and 3'-MGB; the fluorescent label of pol-5'p is 5'-TAMRA and 3'-BHQ2; the fluorescent label of pol-3'p is 5'-Cy5.5 and 3'-BHQ3; the fluorescent label of env-1p is 5'-VIC and 3'-MGB; the fluorescent label of env-2p is 5'-Unlabeled and 3'-MGB; the fluorescent label of env-3p is 5'-VIC and 3'-MGB; the fluorescent label of RPP30-1p is 5'-ROX and 3'-BHQ2; the fluorescent label of RPP30-2p is 5'-Cy5 and 3'-BHQ3.

2. The method according to claim 1, characterized in that The kit also includes control substances, which include negative control substances and positive control substances; the negative control substance is nuclease-free water, and the positive control substance is a fragmented plasmid standard substance; the kit also includes a digital PCR amplification enzyme premix and a fluorescein sodium salt solution, and the digital PCR amplification enzyme premix includes dNTPs, Taq enzyme and PCR buffer.

3. The method according to any one of claims 1 to 2, characterized in that: The PCR reaction system is 25 μL, and / or the PCR reaction procedure corresponding to the PCR chip is as follows: Droplet generation step: temperature 40°C; Pre-denaturation step: temperature 95°C, time 5 minutes; PCR reaction step: denaturation temperature 95°C for 15 seconds, annealing temperature 60°C for 30 seconds, wherein the PCR reaction step is cycled multiple times; Pressure release step: temperature 25°C.

4. The method according to claim 3, characterized in that The positive droplets containing different color fluorescent signals after the digital PCR test and the integrity of HIV-1 nucleic acid are shown in the following table:

5. The method according to claim 4, characterized in that After the digital PCR detection, the positive droplets containing different color fluorescence signals all have corresponding fluorescence values. The specific fluorescence threshold and fluorescence compensation parameters are substituted into the fluorescence value to obtain the number of positive droplets in each fluorescence channel and the corresponding copy number concentration of each target nucleic acid, and the data is analyzed according to the following formula:

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