A multiplex primer combination for detecting HIV-1 drug-resistant gene mutations and application thereof

Abstract

The application discloses a multiplex primer combination for HIV-1 drug-resistant gene mutation detection and application thereof. The primer combination comprises first round amplification primers and second round amplification primers; the nucleic acid sequence of the first round amplification primers comprises sequences shown in SEQ ID NO. 1-8; and the nucleic acid sequence of the second round amplification primers comprises sequences shown in SEQ ID NO. 9-16. The application uses specific primer combinations in the form of multiplex nested PCR, reduces the target region amplification length, improves the amplification capacity, and is more simple to operate, and has higher sensitivity and amplification efficiency (samples with CT values higher than 35 can still be effectively amplified) compared with traditional long fragment amplification.

Description

Technical Field

[0001] This invention belongs to the fields of HIV drug resistance gene detection and multiplex PCR, and relates to a multiplex primer combination for detecting HIV-1 drug resistance gene mutations and its application. Background Technology

[0002] HIV infection leading to a CD4 cell count below 200 μL is known as the AIDS stage, which carries a high mortality rate. In recent years, thanks to the application of antiretroviral therapy (ART), the life expectancy of AIDS patients who successfully receive ART has become close to that of uninfected individuals. However, this therapy is affected by HIV drug resistance gene mutations, making the detection of HIV drug resistance gene mutation sites increasingly important.

[0003] HIV drug resistance gene mutation sites are mainly distributed in the reverse transcriptase, protease, and integrase coding regions of the POL gene in its genome. Currently, many PCR detection technologies detect drug resistance by amplifying each enzyme region individually and then performing Sanger sequencing. Although this method can detect drug resistance segments, the amplification process is cumbersome and the amplified fragments are long, which can lead to insufficient amplification sensitivity and miss samples with low viral load.

[0004] In addition, while the traditional Sanger sequencing method for detecting drug resistance genes has high sequencing accuracy, it is limited by the sequencing principle of its read peaks. This technology has low sensitivity for detecting low-frequency mutations (below 20%), which greatly affects the ability to detect mixed strain infection samples.

[0005] Therefore, there is an urgent need to provide a primer combination and method that can detect low-frequency mutations in HIV-1 drug resistance genes. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a multiple primer combination for detecting HIV-1 drug resistance gene mutations and its application. Through design and practice, a novel HIV drug resistance gene detection technology is proposed. By combining a novel primer design with NGS, the amplification efficiency of HIV drug resistance target regions can be maximized, and the detection of low-frequency drug resistance mutations can be achieved. This is of great significance for guiding medication use in HIV-infected patients in clinical practice.

[0007] To achieve this objective, the present invention employs the following technical solution:

[0008] In a first aspect, the present invention provides a primer combination for detecting HIV-1 drug resistance gene mutations, the primer combination comprising a first-round amplification primer and a second-round amplification primer; the nucleic acid sequence of the first-round amplification primer comprises the sequences shown in SEQ ID NO. 1-SEQ ID NO. 8; the nucleic acid sequence of the second-round amplification primer comprises the sequences shown in SEQ ID NO. 9-SEQ ID NO. 16.

[0009] This invention uses multiple nested PCR with specific primer combinations to reduce the target region amplification length and improve amplification capacity. Compared with traditional long fragment amplification, it has higher sensitivity and amplification efficiency (samples with CT values ​​higher than 35 can still be effectively amplified), and the operation is simpler.

[0010] SEQ ID NO. 1: GACCAGAGCCAACAGCC.

[0011] SEQ ID NO. 2:ACTGGTACAGTTTCAATAGGACT.

[0012] SEQ ID NO. 3: GGATGGCCCAAARGTTAAACAAT.

[0013] SEQ ID NO. 4: ATAACTATGTCTGGATTTTGTTTTCTAAAAGG.

[0014] SEQ ID NO. 5: GTAGCATGACAAAAATCTTAGAGCCY.

[0015] SEQ ID NO. 6: TGTATRTCATTGACAGTCCA.

[0016] SEQ ID NO. 7: TATCACAGYAATTGGAGAGCAATGG.

[0017] SEQ ID NO. 8: GCCACACAATCAKCACCT.

[0018] SEQ ID NO. 9: GAAGAMATRAATTTGCCAGGAA.

[0019] SEQ ID NO. 10: ACAGTTTCAATAGGACTAATKGG.

[0020] SEQ ID NO. 11: GTTAAACAATGGCCATTGACAG.

[0021] SEQ ID NO. 12: TATTGCTGGTGATCCTTTCCATCC.

[0022] SEQ ID NO. 13: GTAGCATGACAAAAATCTTAGAGCCY.

[0023] SEQ ID NO. 14: GCTGKACTGTCATTTRTCAGGA.

[0024] SEQ ID NO. 15: GCYAGCTGTGATAAATGTCARCTA.

[0025] SEQ ID NO. 16: TGCCATCTGTTTTCCATARTCC.

[0026] In this system, the degenerate base Y represents C / T, R represents A / G, K represents G / T, and M represents A / C.

[0027] Preferably, the HIV-1 drug resistance gene includes the pol gene.

[0028] Preferably, the mutation site of the drug resistance gene mutation is any one or a combination of at least two of the mutation sites shown in Table 1 below.

[0029] Table 1

[0030]

[0031]

[0032] In a second aspect, the present invention provides the application of the primer combination described in the first aspect for detecting HIV-1 drug resistance gene mutations in the preparation of products for detecting HIV-1 drug resistance gene mutations.

[0033] Thirdly, the present invention provides a kit for detecting HIV-1 drug resistance gene mutations, the kit comprising the primer combination for detecting HIV-1 drug resistance gene mutations as described in the first aspect.

[0034] Fourthly, the present invention provides the application of the primer combination described in the first aspect for detecting HIV-1 drug resistance gene mutations in the detection of HIV-1 drug resistance gene mutations.

[0035] Fifthly, the present invention provides a method for detecting HIV-1 drug resistance gene mutations, the method comprising the following steps:

[0036] (1) Collect nucleic acid from HIV-1 infected samples and detect the CT value of HIV-1 by real-time PCR;

[0037] (2) Reverse transcription of the entire genome of the RNA virus was performed on the nucleic acid of HIV-1 infected samples to obtain cDNA;

[0038] (3) The primer combination for detecting HIV-1 drug resistance gene mutations described in the first aspect is used to perform the first round of amplification and the second round of amplification. After the second round of amplification is completed, the fragments of the second round of amplification products are detected by Q-seq100 or agarose gel electrophoresis.

[0039] (4) The second-round amplification products were purified using purification magnetic beads and the nucleic acid was quantified using Qubit4.0;

[0040] (5) Library construction using endonucleases or transposases;

[0041] (6) Perform SE100 single-end NGS sequencing on the library sample set;

[0042] (7) Filter and quality control the data after the machine is run, extract the sequencing depth and coverage of the HIV-1 genome, and use the Stanford University HIV drug resistance gene mutation site database as a reference to analyze the drug resistance mutation sites of the sample nucleic acid.

[0043] Preferably, the amplification system for the first round of amplification in step (3) includes: cDNA obtained by reverse transcription of the whole genome of an HIV-1 infected sample, primers for the first round of amplification, and amplification reaction solution.

[0044] Preferably, the nucleic acid sequence of the primers for the first round of amplification includes the sequences shown in SEQ ID NO.1-SEQ ID NO.8.

[0045] Preferably, the amplification system for the second round of amplification in step (3) includes: the product obtained from the first round of amplification, the primers for the second round of amplification, and the amplification reaction solution.

[0046] Preferably, the nucleic acid sequence of the second round of amplification primers includes the sequences shown in SEQ ID NO.9-SEQ ID NO.16.

[0047] Preferably, the reaction conditions for the first and second rounds of amplification in step (3) are each independently: (1) 96-98℃, 5-8min, 1-3 cycles; (2) 96-98℃, 20-30s, 56-58℃, 20-30s, 72-74℃, 1-2min, 30-35 cycles; (3) 72-74℃, 5-8min, 1-3 cycles; (4) 4℃ hold.

[0048] The specific point values ​​in the above 96-98℃ range can be selected as 96℃, 97℃, 98℃, etc.

[0049] The specific time values ​​in the above 5-8 min range can be selected as 5 min, 6 min, 7 min, 8 min, etc.

[0050] The specific point values ​​in the above 1-3 loops can be selected as 1 loop, 2 loops, 3 loops, etc.

[0051] The specific point values ​​in the above 20-30 s can be selected as 20s, 21s, 22s, 24s, 26s, 28s, 29s, 30s, etc.

[0052] The specific point values ​​in the above 56-58℃ range can be selected as 56℃, 57℃, 58℃, etc.

[0053] The specific point values ​​in the above 72-74℃ range can be selected as 72℃, 73℃, 74℃, etc.

[0054] The specific point values ​​in the above 1-2 min range can be selected as 1 min, 1.5 min, 2 min, etc.

[0055] The specific point values ​​in the above 30-35 cycles can be selected as 30 cycles, 31 cycles, 32 cycles, 33 cycles, 34 cycles, 35 cycles, etc.

[0056] Preferably, the library construction in step (5) includes nucleic acid fragmentation, nucleic acid fragmentation termination, and PCR.

[0057] Preferably, the HIV-1 infected sample in step (1) includes blood or plasma.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] (1) Compared with traditional single-target amplification, this invention reduces the target amplification length and improves amplification capacity by using multiple nested PCR. Compared with traditional long fragment amplification, it has higher sensitivity and amplification efficiency (samples with CT values ​​higher than 35 can still be effectively amplified), and the operation is simpler.

[0060] (2) Compared with traditional Sanger sequencing, this invention, combined with high-throughput sequencing technology, achieves a detection limit of 5% for low-frequency mutations, which can better address mixed infection samples of multiple strains, correctly guide clinical medication for patients, and improve patient survival rate. Attached Figure Description

[0061] Figure 1 Distribution map of primers designed for the HIV genome;

[0062] Figure 2A The graph shows the qPCR amplification curve and CT value results.

[0063] Figure 2BThe result of Q-seq100 is shown in the figure;

[0064] Figure 3 This is a graph showing the sequencing depth results;

[0065] Figure 4 This is a Qseq-100 fragment detection image;

[0066] Figure 5 This is a peak diagram of Qseq-100 amplification.

[0067] Figure 6 This is for comparison with the sequencing peak diagram. Detailed Implementation

[0068] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0069] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0070] Example 1

[0071] This embodiment provides a method for detecting HIV-1 drug resistance gene mutations.

[0072] The HIV-1 pseudovirus (FNRV2708) from Fubai Ao (Suzhou) Biomedical Technology Co., Ltd. was used as a template for amplification testing. The amplification primers and quantitative PCR sequences are shown in Table 1, the main reagents are shown in Table 2, the multiplex nested PCR reaction conditions are shown in Table 3, the first round PCR reaction system is shown in Table 4, and the second round PCR reaction system is shown in Table 5.

[0073] The specific steps are as follows: The HIV-1 pseudovirus was converted from 10... 8 Dilute to 10 5 Nucleic acid extraction was performed after the exponentiation was increased. The extraction reagent used was the nucleic acid extraction kit (KS619-DNAmN24) from Jinqirui Biotechnology Co., Ltd., to extract and obtain HIV-1 pseudovirus nucleic acid. The viral nucleic acid was diluted in five gradients: 1×, 2×, 8×, 32×, and 128×. Quantitative PCR using dye-based methods was then performed. The fluorescence quantitative PCR reagent was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd. Commercially available SYBR and GREEN fluorescence quantitative PCR reagents can be used as alternatives.

[0074] All gradient pseudoviral nucleic acids were reverse transcribed using the full-length cDNA synthesis kit (13488) from Yisheng Biotechnology (Shanghai) Co., Ltd. The resulting cDNA was then amplified in the first round using multiplex amplification reaction solution (BK0051) from Aibote Biotechnology (Wuhan) Co., Ltd. The primer sequences used were SEQ ID NO.1-SEQ ID NO.8. The amplification system is shown in Table 5, and the amplification program is shown in Table 4. After the first round of amplification, the amplified products were used as a template to prepare the reaction system as shown in Table 6, and the amplification program was shown in Table 4. The second round of amplification was then performed, and the resulting products were detected by Q-seq100 fragment analysis.

[0075] During the library construction stage, various commercial library construction kits can be used for library preparation. In this example, the transposase rapid library preparation kit (TD501) from Novizan (Nanjing) Biotechnology Co., Ltd. was used to prepare a library from the amplicon of the lowest gradient nucleic acid and then sequenced it.

[0076] Table 2

[0077]

[0078]

[0079] Table 3

[0080] reagent components use Multiplex amplification reaction solution Multiple nested PCR reaction reverse transcriptase Viral whole genome reverse transcription

[0081] Table 4

[0082]

[0083] Table 5

[0084] Components Volume (μL) Reverse transcription of cDNA 1 First round of amplification primer mix 4 (Final concentration of each primer: 0.1 μM) Amplification reaction solution 12.5 Nuclease-free water 7.5

[0085] Table 6

[0086] Components Volume (μL) First round PCR products 1 Second round of amplification primers 4 (Final concentration of each primer: 0.1 μM) Amplification reaction solution 12.5 Nuclease-free water 7.5

[0087] Results: The Q-seq100 results for the 5 gradients are as follows Figure 2B As shown, each gradient exhibits four distinct peaks, corresponding to the products of the four target regions, with sizes of approximately 250bp / 350bp / 420bp / 730bp, which are roughly close to the corresponding lengths on the HIV-1 genome (though there are some differences between different HIV strains). Each gradient amplifies distinct bands, and the qPCR amplification curves and CT values ​​for each gradient are shown below. Figure 2A As shown.

[0088] The sequencing depth results after library construction with the lowest gradient (128×) nucleic acid amplicon are as follows: Figure 3As shown, with a sequencing data volume of 0.3M, the average target depth is greater than 10000×, which is higher than the minimum depth (100×) required for drug resistance analysis.

[0089] Example 2

[0090] In this embodiment, the primer sequences provided in Table 2 are used as a control, and several primer pairs are replaced for amplification.

[0091] The samples used were reverse-transcribed cDNA from the HIV-1 pseudoviral nucleic acid (128×) gradient in Example 1. The amplification and reaction system were consistent with Example 1. The primer sequences for the first and second rounds of amplification in the control group were consistent with those in Example 1. In test group 1, SEQ ID NO.1 and SEQ ID NO.6 were replaced with SEQ ID NO.19 and SEQ ID NO.20 in Table 7. In test group 2, SEQ ID NO.3 and SEQ ID NO.8 were replaced with SEQ ID NO.20 and SEQ ID NO.21 in Table 7. In test group 3, only SEQ ID NO.1 and SEQ ID NO.8 were used in the first round of amplification, and only SEQ ID NO.9 and SEQ ID NO.16 were used in the second round of amplification for long fragment amplification. In test group 4, SEQ ID NO.9 and SEQ ID NO.16 were replaced with SEQ ID NO.23 and SEQ ID NO.24 in Table 7. In test group 5, SEQ ID NO.3 and SEQ ID NO.8 were replaced with SEQ ID NO.25 and SEQ ID NO.26 in Table 7.

[0092] Results: Qseq-100 fragment detection images are shown below. Figure 4 As shown, the peak diagram of the control group fragment is complete, and the bands of the four target regions are clear; the peak diagram of test group 1 shows that only target region 2 is amplified, and the other three target regions have no obvious peaks; the peak diagram of test group 2 shows that the amplification efficiency of target region 3 is significantly reduced; in test 3, which involves long fragment amplification, the drug-resistant target region is not significantly amplified; in test group 4, target region 3 can amplify a band, but its amplification efficiency is significantly reduced; in test group 5, target regions 3 and 4 are not amplified.

[0093] Table 7

[0094]

[0095] Example 3

[0096] The difference between this embodiment and Embodiment 1 is that long-fragment amplification of the nucleic acid of HIV-1 infected samples is performed.

[0097] This example used nucleic acid samples from 7 HIV-1 infected samples, provided by Guangzhou KingMed Diagnostics Center Co., Ltd. The reverse transcription and amplification procedures were the same as in Example 1, and HIV-1 pseudovirus nucleic acid was added as a quality control in the test.

[0098] Results: The Qseq-100 amplification peak diagram is as follows: Figure 5 As shown, the fake virus control sample and the 7 HIV-1 infected samples all had four peaks matching the target size, and the peak diagrams showed no obvious primer dimers or non-specific amplification.

[0099] Example 4

[0100] The difference between this embodiment and Embodiment 1 is that Sanger detection is performed.

[0101] The amplification products of the seven samples from Example 3 were subjected to NGS and Sanger assays, respectively. The NGS library preparation process was the same as described in Example 1, and the Sanger assay was provided by Guangzhou Aiji Biotechnology Co., Ltd. After sequencing, the drug resistance detection results were compared.

[0102] Results: The results of NGS and Sanger drug resistance detection were compared and are shown in Table 8 below. In the 7 samples, the drug resistance site mutations detected by Sanger were also shown in the drug resistance results of NGS. In addition, for low-frequency mutations such as 48V in HIVS589, Sanger missed them and no overlapping peaks were found in the sequencing peak diagram.

[0103] In HIVS519 sample, NGS resistance analysis revealed a 163K mutation with a mutation frequency of 42%, but the Sanger results did not show the mutation. This was confirmed by comparing the sequencing peak patterns. Figure 6 It was found that the base mutation at this site appeared as a duplicate peak in the sequencing peak diagram, which resulted in a missed detection in the drug resistance mutation results. This is a situation that Sanger sequencing cannot avoid.

[0104] Table 8

[0105]

[0106]

[0107] In summary, this invention uses a multiplex nested PCR method with specific primer combinations to reduce the target region amplification length and improve amplification capacity. Compared with traditional long-fragment amplification, it has higher sensitivity and amplification efficiency (samples with CT values ​​higher than 35 can still be effectively amplified), and the operation is simpler.

[0108] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A primer combination for detecting HIV-1 drug-resistant gene mutation, characterized by, The primer combination comprises first round amplification primers and second round amplification primers; the nucleic acid sequence of the first round amplification primers comprises the sequences shown in SEQ ID NO. 1-SEQ ID NO. 8; the nucleic acid sequence of the second round amplification primers comprises the sequences shown in SEQ ID NO. 9-SEQ ID NO.

16.

2. The primer combination for detecting the drug-resistant genetic mutation of HIV-1 according to claim 1, characterized in that, The HIV-1 drug resistance gene comprises a pol gene.

3. Use of the primer combination for detecting mutations of HIV-1 drug resistance genes according to claim 1 or 2 in the preparation of a product for detecting mutations of HIV-1 drug resistance genes.

4. A kit for detecting a drug-resistant genetic mutation of HIV-1, characterized by, The kit comprises the primer combination for detecting mutations of HIV-1 drug resistance genes according to claim 1 or 2.

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