A primer set and method for targeted library construction of HIV-1 drug-resistant sequences

Abstract

The present invention provides a primer set and method for targeted library construction of HIV-1 drug-resistant sequences, the primer set includes amplification primers, and the nucleotide sequences of the amplification primers are shown in SEQ ID NO.1-38; a method for targeted library construction and detection of HIV-1 drug-resistant sequences, including extracting HIV-1 nucleic acid samples; a first round of multiple PCR reaction, and magnetic bead purification after the reaction to obtain multiple PCR products; a second round of linker sequence PCR reaction, and magnetic bead purification after the reaction to obtain amplified products; the amplified products are sequenced on a machine, and the sequencing results are analyzed to obtain relevant mutation type information. The targeted amplification library construction of the present invention can capture the target area more accurately, can effectively remove background sequence interference, obtain more effective sequencing data, and reduce sequencing costs and analysis costs; the amplification length is shorter, the amplification efficiency is higher, and the success rate is higher. The sequencing method used in the present invention can more accurately detect base sites with low mutation rates, detect drug-resistant mutation sites as low as 1%, and find very early drug resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a primer set and a method for building a library for HIV-1 drug-resistant sequences in a targeted manner. Background Art

[0002] AIDS, or acquired immunodeficiency syndrome, is a chronic and fatal infectious disease caused by human immunodeficiency virus (HIV), which is divided into human immunodeficiency virus type 1 (HIV-1) and human immunodeficiency virus type 2 (HIV-2). HIV-1 is the main strain currently prevalent in the world. Data show that among infected people who receive ART treatment, 10%-30% of patients will experience viral rebound after treatment, and the proportion of infected people who fail to use first-line treatment is 52.3% with drug resistance. Therefore, it is of great significance to test patients for drug resistance.

[0003] Currently, ART treatment cannot be discontinued, and HIV RNA can be reduced to an undetectable level during treatment. However, due to the existence of HIV DNA viral reservoirs, HIV is still incurable. During the continuous use of ART treatment, there is a high risk of drug resistance, and the patient will be in a state of low viremia in the early stages. In the state of low viremia, the HIV RNA viral load is extremely low, and it is difficult to detect successfully using existing drug resistance detection methods. Although the success rate of HIV RNA drug resistance detection can be improved through methods such as viral enrichment, the enrichment process is still relatively complicated and requires the use of viral enrichment reagents or high-speed centrifuges. In addition, the viral load cannot be detected, which means that drug resistance cannot be detected and predicted.

[0004] At present, the drug resistance detection method of PCR plus Sanger sequencing used in the prior art cannot detect drug resistance mutation sites below 20%, and the detection limit is relatively high, generally requiring the sample load to be above 1000 copies / ml, so there are often cases of missed detection and failure to detect very early drug resistance. Based on this, the present invention provides a new primer set and method for targeted library construction of HIV-1 drug resistance sequences. Summary of the invention

[0005] Based on the above description, the present invention provides a primer set and method for targeted library construction of HIV-1 drug-resistant sequences. The targeted library construction method is used to amplify the drug-resistant region sequence of HIV RNA or DNA, and the second-generation sequencing method is used to obtain sequence information.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In a first aspect, the present invention provides a primer set for targeted library construction of HIV-1 drug-resistant sequences, the primer set comprising amplification primers, the nucleotide sequences of the amplification primers are shown in SEQ ID NOs. 1 to 28.

[0008] In a second aspect, the present invention provides use of the primer set described in the first aspect in targeted library construction of HIV-1 drug-resistant sequences.

[0009] In a third aspect, the present invention provides use of the primer set as described in the first aspect in preparing a kit for detecting HIV-1 drug resistance sequences.

[0010] In a fourth aspect, the present invention provides a method for targeted library construction and detection of HIV-1 drug-resistant sequences for non-disease diagnosis and treatment purposes, comprising the following steps:

[0011] S1. Extract HIV-1 nucleic acid samples;

[0012] S2, preparing a reaction system with a PCR amplification enhancer, primers, HIV-1 nucleic acid sample and amplification enzyme, performing the first round of multiplex PCR reaction according to the procedure, and performing magnetic bead purification after the reaction to obtain multiplex PCR products;

[0013] S3, preparing the purified multiple PCR products, enhancer, linker reagent and enzyme mixture into an amplification reaction system, performing the second round of linker sequence PCR reaction according to the procedure, and performing magnetic bead purification after the reaction to obtain the amplified product;

[0014] S4. The amplified products are sequenced on a machine, and the sequencing results are analyzed to obtain relevant mutation type information.

[0015] Based on the above technical solution, the present invention can also be improved as follows.

[0016] Furthermore, the step S1 specifically includes the following steps:

[0017] S11, take a blood sample, add a premixed solution of buffer GB and proteinase K to the sample, and mix by inversion;

[0018] S12, place at room temperature, add buffer BD, and mix by inversion;

[0019] S13, adding the solution and flocculent precipitate obtained in step S12 to an adsorption column CG2, centrifuging, discarding the waste liquid, and placing the adsorption column CG2 in a collection tube;

[0020] S14, adding buffer GDB to the adsorption column CG2, centrifuging, discarding the waste liquid, and placing the adsorption column CG2 in a collection tube;

[0021] S15, add rinsing liquid to the adsorption column CG2, centrifuge, discard the waste liquid, place the adsorption column CG2 in the collection tube, repeat this round of steps, centrifuge, discard the waste liquid, and place the adsorption column CG2 at room temperature to completely dry the residual rinsing liquid in the adsorption material;

[0022] S16. Transfer the adsorption column CG2 into a centrifuge tube, add elution buffer TB to the middle of the adsorption membrane, place it at room temperature, centrifuge it, and collect the solution into the centrifuge tube. The solution in the tube is the extracted HIV-1 nucleic acid sample.

[0023] Furthermore, the method for targeted library construction and detection of HIV-1 drug-resistant sequences is characterized in that step S2 specifically comprises the following steps:

[0024] S21, taking out the PCR amplification enhancer NB, primers and amplification enzyme in the kit, thawing them, vortexing and centrifuging them instantaneously for later use;

[0025] S22, taking out the PCR amplification enhancer M and melting it at room temperature;

[0026] S23, prepare the reaction system: PCR amplification enhancer NB 3.5uL, PCR amplification enhancer M 2.5uL, primer 5uL, sample 5uL, amplification enzyme 10uL, add sterile water to 30uL;

[0027] S24, performing PCR amplification according to the amplification program;

[0028] S25, purify the amplified product with magnetic beads.

[0029] Further, in step S24, the amplification program is: heating to 105°C; 95°C for 3 min 30 s; 98°C for 40 s, 55°C for 1 min, 60°C for 1 min, 65°C for 1 min, 18 cycles; 72°C for 5 min.

[0030] Furthermore, the step S3 specifically includes the following steps:

[0031] S31. Prepare the reaction system according to the following table: 13.5uL of the purified multiplex PCR product in step S2, 2.5uL of enhancer M, 1uL of linker reagent, 10uL of enzyme mixture, 3uL of sterile water, and a total system of 30uL.

[0032] S32, performing PCR amplification according to the amplification program; the amplification program is: heating to 105°C; 95°C for 3min30s; 98°C for 20s, 58°C for 1min, 72°C for 30s, 9 cycles; 72°C for 5min;

[0033] S33, purify the amplified product with magnetic beads.

[0034] Furthermore, the step S4 specifically includes:

[0035] The amplified product is sequenced on a second-generation sequencing machine to obtain the sequencing results;

[0036] The sequencing results are spliced ​​and analyzed using standardized bioinformatics splicing and analysis system software to obtain complete drug resistance sequences and mutation rate data, which are then imported into the HIV drug resistance database to obtain relevant mutation type information.

[0037] Compared with the prior art, the technical solution of this application has the following beneficial technical effects:

[0038] The primer set, kit and library construction detection method for targeted library construction of HIV-1 drug resistance sequences provided by the present invention are simple, convenient and fast to operate. In particular, the targeted library construction method is used to amplify the drug resistance region sequence of HIV RNA or DNA, and the second generation sequencing method is used to obtain sequence information.

[0039] The advantages of the present invention are as follows:

[0040] 1. Compared with the common reverse transcription library construction method, targeted amplification library construction can capture the target area more accurately, effectively remove background sequence interference, obtain more effective sequencing data, and reduce sequencing and analysis costs.

[0041] 2. Compared with the method of drug resistance detection by PCR plus Sanger sequencing, the amplification success rate of the present invention is higher. PCR plus Sanger sequencing usually directly amplifies the entire drug resistance sequence, with a length of about 1500bp; while the length of the fragment amplified by the present invention is only about 175bp, the amplification length is shorter, the amplification efficiency is higher, and the success rate is also higher.

[0042] 3. The library construction method proposed in the present invention can be used for HIV DNA sequencing library construction even in patients with undetectable viral load (HIV RNA drug resistance detection cannot be performed), thereby improving the success rate of detection.

[0043] 4. Compared with the Sanger sequencing method, the second-generation sequencing method can more accurately detect base sites with low mutation rates, can detect drug-resistant mutation sites as low as 1%, and can discover extremely early drug resistance. DETAILED DESCRIPTION

[0044] The implementation method of this solution will be further described in detail below. Obviously, the described embodiments are only part of the embodiments of this solution, rather than an exhaustive list of all the embodiments. It should be noted that the embodiments and features in the embodiments of this solution can be combined with each other without conflict.

[0045] The contents disclosed in the present invention can be improved in terms of materials, methods and reaction conditions at the same time, and all these improvements should fall within the spirit and scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The reagents and biological materials, unless otherwise specified, can be obtained from commercial channels; the databases used are all public online databases.

[0046] Example 1

[0047] This example describes the nucleotide sequences of amplification primers, as shown in SEQ ID NOs. 1-38.

[0048]

[0049]

[0050] Example 2

[0051] This example describes a method for targeted library construction and detection of HIV-1 drug-resistant sequences, which is not intended for disease diagnosis and treatment.

[0052] It should be noted that: this example uses a blood genomic DNA extraction kit (DP348) produced by Tiangen Company. The kit instructions are followed to extract at least 200 μL of blood sample and collect nucleic acids with 50-80 μL of eluent.

[0053] The specific steps include:

[0054] Step S1, extracting HIV-1 nucleic acid samples.

[0055] Specifically, S11, take 200 μL of blood sample, add 200 μL of buffer GB and 20 μL of premixed solution of Proteinase K to the sample, mix thoroughly by inversion, and place at 56° C. for 10 min, during which time the sample is mixed by inversion several times.

[0056] S12. Leave at room temperature for 2 to 5 minutes, add 350 μL buffer BD, and mix by inversion.

[0057] S13. Add the solution and flocculent precipitate obtained in step S12 into an adsorption column CG2 (the adsorption column CG2 is placed in a collection tube), centrifuge at 12000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CG2 in the collection tube.

[0058] S14. Add 500 μL of buffer GDB to the adsorption column CG2, centrifuge at 12000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CG2 in the collection tube.

[0059] S15. Add 600 μL of rinsing solution PWB to the adsorption column CG2 (please check whether anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CG2 in the collection tube; repeat this round of steps once. Centrifuge at 12000 rpm for 2 minutes, pour out the waste liquid, and place the adsorption column CG2 at room temperature for 2 minutes to completely dry the residual rinsing solution in the adsorption material.

[0060] S16. Transfer the adsorption column CG2 into a 1.5 ml centrifuge tube, add 50-80 μL of elution buffer TB to the middle of the adsorption membrane, let it stand at room temperature for 2 minutes, centrifuge at 12000 rpm for 2 minutes, collect the solution into the centrifuge tube, and the solution in the tube is the extracted HIV-1 nucleic acid sample.

[0061] Step S2, first round of multiplex PCR reaction: prepare a PCR amplification enhancer, primers, HIV-1 nucleic acid sample and amplification enzyme into a reaction system, perform the first round of multiplex PCR reaction according to the procedure, and perform magnetic bead purification after the reaction to obtain multiplex PCR products.

[0062] Specifically, S21, take out the PCR amplification enhancer NB, primers and amplification enzyme in the kit from the -20°C refrigerator, place them on an ice box to melt, vortex briefly to mix and centrifuge them instantaneously after melting, and place them on an ice box for later use.

[0063] S22. Take out the PCR amplification enhancer M from the 4°C refrigerator and melt it at room temperature.

[0064] S23. Prepare the reaction system on an ice box according to the following table:

[0065] PCR amplification enhancer NB 3.5uL, PCR amplification enhancer M 2.5uL, primer 5uL, sample 5uL, amplification enzyme 10uL, add sterile water to make up to 30uL.

[0066] S24. Perform PCR amplification according to the amplification program.

[0067] The amplification procedure is:

[0068]

[0069] S25, purify the amplified product with magnetic beads.

[0070] Specifically include:

[0071] (1) Take out the purified magnetic beads from the 4°C refrigerator in advance, mix them and place them at room temperature for 30 minutes; vortex the purified magnetic beads that have been equilibrated to room temperature and set aside.

[0072] (2) Add 0.9 times the volume of magnetic beads (27 μL) to the 30 μL reaction system after amplification, pipette or vortex to mix, and let stand at room temperature for 5 min.

[0073] (3) Centrifuge briefly and place the PCR tube on a magnetic rack for 3 min to allow the solution to clarify.

[0074] (4) Completely remove the supernatant, remove the PCR tube from the magnetic rack, add 50 μL of YF buffer B to the tube, pipette to mix, and let stand at room temperature for 5 min.

[0075] (5) Centrifuge briefly and place the PCR tube on a DynaMag-96Side magnetic stand for 3 min.

[0076] (6) Keep the PCR tube on the magnetic rack, carefully remove the supernatant, add 180 μL of 80% ethanol solution to the PCR tube, and let it stand for 30 seconds.

[0077] (7) Keep the PCR tube on the magnetic rack, discard the supernatant, add 180 μL of 80% ethanol solution to the PCR tube again, let it stand for 30 seconds, and discard the supernatant.

[0078] (8) Cover the tube and centrifuge briefly to remove the remaining ethanol to the bottom of the tube. Place the PCR tube on a magnetic rack and carefully use a 10 μL pipette to remove the remaining ethanol at the bottom, taking care not to absorb the magnetic beads.

[0079] (9) Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3 to 5 minutes to dry the magnetic beads and allow the residual ethanol to evaporate completely.

[0080] (10) Add 24 μL of Nuclease-Free Water, remove the PCR tube from the magnetic rack, pipette or vortex to mix, and let stand at room temperature for 2 min.

[0081] (11) Centrifuge briefly and place the PCR tube on a magnetic rack for 2 min to allow the solution to clarify.

[0082] (12) Use a pipette to aspirate 13.5 μL of supernatant and transfer it to a new PCR tube. The supernatant in the tube is the purified multiplex PCR product. Mark it and prepare for the subsequent step S3 reaction.

[0083] Step S3, preparing the purified multiple PCR products, enhancer, linker reagent and enzyme mixture into an amplification reaction system, performing the second round of linker sequence PCR reaction according to the procedure, and obtaining the amplified product after magnetic bead purification after the reaction.

[0084] Specifically, S31, prepare the reaction system on an ice box according to the following table: 13.5uL of the purified multiple PCR product in step S2, 2.5uL of enhancer M, 1uL of linker reagent, 10uL of enzyme mixture, 3uL of sterile water, and a total system of 30uL.

[0085] S32, performing PCR amplification according to an amplification program; the amplification program is:

[0086]

[0087] S33, purifying the amplified product with magnetic beads. The process of magnetic bead purification refers to the above step S25 and will not be repeated here.

[0088] Step S4: sequence the amplified product on a machine, analyze the sequencing results, and obtain relevant mutation type information.

[0089] Specifically, it includes: sequencing the amplified product on a next-generation sequencing (NGS) machine to obtain sequencing results;

[0090] The sequencing results are spliced ​​and analyzed using standardized bioinformatics splicing and analysis system software to obtain complete drug resistance sequences and mutation rate data, which are then imported into the HIV drug resistance database to obtain relevant mutation type information.

[0091] Specifically, the FASTQ file of the sequencing result is imported into the HIV DB drug resistance database and converted into a CodFreq file. The conversion parameters are set according to the requirements, and it is recommended to use the default values. Then the CodFreq file is imported into the HIV DB drug resistance database to obtain the relevant mutation type information.

[0092] The embodiments of the present invention have the following advantages:

[0093] 1. Compared with the common reverse transcription library construction method, targeted amplification library construction can capture the target area more accurately, effectively remove background sequence interference, obtain more effective sequencing data, and reduce sequencing and analysis costs.

[0094] 2. Compared with the method of drug resistance detection by PCR plus Sanger sequencing, the amplification success rate of the present invention is higher. PCR plus Sanger sequencing usually directly amplifies the entire drug resistance sequence, with a length of about 1500bp; while the length of the fragment amplified by the present invention is only about 175bp, the amplification length is shorter, the amplification efficiency is higher, and the success rate is also higher.

[0095] 3. The library construction method proposed in the present invention can be used for HIV DNA sequencing library construction even in patients with undetectable viral load (HIV RNA drug resistance detection cannot be performed), thereby improving the success rate of detection.

[0096] 4. Compared with the Sanger sequencing method, the second-generation sequencing method can more accurately detect base sites with low mutation rates, can detect drug-resistant mutation sites as low as 1%, and can discover extremely early drug resistance.

[0097] To further verify the above effects, the following are some cases where the detection success rate is improved:

[0098]

[0099] By testing the above 4 samples that failed HIV RNA resistance detection using the first-generation sequencing method, it was found that 2 samples (T230420002 and T230521003) that failed amplification due to low virus load and 2 samples (T230519003 and H1E387G100QR) that failed detection due to mutations could be successfully detected using the method of the present invention.

[0100] The following are some examples of early detection of drug resistance:

[0101]

[0102] By testing 13 samples that had been tested for HIV RNA resistance using the first-generation sequencing method, this method was used to detect 3 early mutations, namely T230715005, H1E387G101J9, and M21001920636, accounting for 23.1%, showing that the method of the present invention has the ability to discover drug-resistant mutation sites at an earlier stage. At the same time, the proportion of mutated bases in the newly discovered mutation sites using the second-generation sequencing was less than 30%, which is consistent with the detection performance of the first-generation sequencing.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a primer set in preparing a kit for detecting HIV-1 drug resistance sequences, characterized in that: The kit comprises a primer set for targeted library construction of HIV-1 drug-resistant sequences, wherein the primer set comprises amplification primers, and the nucleotide sequences of the amplification primers are shown in SEQ ID NOs. 1-38.

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