A method for screening and identifying unique recombinant strains and prevalent recombinant strains of human immunodeficiency virus type 1

Abstract

The present invention discloses a method for screening and identifying unique recombinant strains of human immunodeficiency virus type 1 and prevalent recombinant strains, which comprises a mosaic diagram generated by amplified fragments of HIV-1 prone breakpoint mutation regions, which is compared with the mosaic diagrams of published CRFs, screening out strains with new mosaic diagrams, and establishing a new and efficient full-length gene sequence amplification method for preventing artificial recombination, establishing an evolutionary tree together with the amplified full-length sequence and the reference sequence, comprehensively judging whether it is URFs through homology analysis and mosaic diagrams, and further judging whether it is CRFs based on the number of prevalent strains of the same URFs. This technical method has the characteristics of low cost and high efficiency, and all traditional samples to be selected are subjected to full-length amplification, and are changed to amplification analysis and screening for prone mutation regions, overcoming the risk of artificial recombination caused by multi-fragment amplification of the full length, and increasing the amplification success rate through technical improvements.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to a method for screening and identifying unique recombinant strains and prevalent recombinant strains of human immunodeficiency virus type 1. Background Technology

[0002] Human immunodeficiency virus (HIV) is an RNA virus with high variability, causing acquired immunodeficiency syndrome (AIDS). In recent years, the subtypes circulating globally have become increasingly complex. HIV-1 is highly variable, and at least 10 subtypes (A, B, C, D, F, G, H, J, K) have been identified in HIV-1 group M. When a region has a complex prevalent subtype distribution and reaches a certain level of transmission in the population, recombination between HIV-1 subtypes is likely to occur, producing unique recombinant forms (URFs). When a certain URF tends to become the dominant strain during an epidemic, it becomes a circulating recombinant form (CRF). To date, 132 CRFs have been identified domestically and internationally, and they are dominant in epidemics. HIV-1 gene mutations can easily lead to drug resistance, increasing the pressure on infectious disease prevention and control efforts. Timely screening and identification of new URFs and CRFs are of great significance for monitoring HIV-1 transmission.

[0003] Currently, the most common method for amplifying the HIV-1 gene sequence is to divide the full-length 9K bp HIV-1 gene into two or more segments, amplify each segment separately, and then sequence them. The resulting sequence is then assembled to obtain the full-length sequence. The amplification method typically involves two rounds of amplification: reverse transcription followed by nested PCR. The amplified sequences are then compared to confirm the genotype and whether they are URFs or CRFs. Because HIV-1 is an RNA virus with high variability, it replicates extensively within the same host, resulting in population genomic diversity, often existing as quasi-species. Quasi-species existence means that the virus exists in the same sample as multiple strain gene sequences, and what is amplified or detected is generally the dominant strain or the most prevalent gene fragment within that quasi-species. Therefore, when using multi-segment amplification and assembly for identification of new URFs or CRFs, there is a risk of artificial recombination. This means that different strain fragments are amplified separately, and the resulting gene sequences may originate from different strains within the same sample, rather than from the same strain. The present invention aims to adopt a method for amplifying the full length of HIV genes to avoid the risk of artificial recombination, so that the amplified genes are gene fragments of the same strain.

[0004] Most current literature only performs final discrimination on newly identified URFs or CRFs, without detailing screening methods from large samples. Some literature mentions amplifying full-length sequences for screening, performing full-length amplification and sequencing on all samples. However, full-length sequencing is costly, time-consuming, and labor-intensive, and some literature reports a full-length amplification rate of only 66%. The screening and identification method of this invention first compares the mosaic patterns of different gene regions in a large number of samples, then performs full-length amplification on some suspected new subtypes to narrow down the target range, and finally performs full-length amplification on the target strains and compares them. This screening and identification method and process not only provides a new approach for the screening and discovery of new subtypes, but also reduces economic and time costs and improves the efficiency of new subtype screening. Summary of the Invention

[0005] To address the problems existing in the background art, the present invention provides a method for screening and identifying unique recombinant strains and prevalent recombinant strains of human immunodeficiency virus type 1 (HIV-1). This method reduces the cost of screening unique recombinant strains and prevalent recombinant strains of HIV-1. Instead of performing full-length amplification and sequencing on all candidate samples in the traditional method, this method performs amplification analysis and screening on easily mutated regions, overcoming the risk of artificial recombination caused by multi-fragment full-length amplification. Through technical improvements, the amplification success rate is increased.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides primers for amplifying the full-length HIV-1 gene sequence, comprising an outer primer pair for the first round of full-length gene sequence amplification by nested PCR and an inner primer pair for the second round of full-length gene sequence amplification.

[0008] The first round of full-length gene sequence amplification used the outer primer pair OF and OR, and the second round of full-length gene sequence amplification used the inner primer pair IF and IR.

[0009] The nucleotide sequence of OF is shown in SEQ ID NO.1;

[0010] The nucleotide sequence of OR is shown in SEQ ID NO.2;

[0011] The nucleotide sequence of IF is shown in SEQ ID NO.3;

[0012] The nucleotide sequence of IR is shown in SEQ ID NO.4.

[0013] In a second aspect, the present invention provides primers for amplifying the HIV-1 gene fragments gag, env, and pol, which are prone to breakpoint mutations, including primer pairs for the first round of nested PCR amplification and primer pairs for the second round of amplification; the primers for the first round of nested PCR amplification of the three gene fragment regions all use the outer primers OF and OR for amplification of the full-length gene sequence; in the primer pairs for the second round of nested PCR amplification, the amplification primers for the gag, env, and pol gene fragments correspond to GF and GR, EF and ER, and PF and PR, respectively;

[0014] The nucleotide sequence of GF is shown in SEQ ID NO.5;

[0015] The nucleotide sequence of GR is shown in SEQ ID NO.6;

[0016] The nucleotide sequence of EF is shown in SEQ ID NO.7;

[0017] The nucleotide sequence of ER is shown in SEQ ID NO.8;

[0018] The nucleotide sequence of PF is shown in SEQ ID NO.9;

[0019] The nucleotide sequence of PR is shown in SEQ ID NO.10.

[0020] Specifically, the nucleotide sequences of OF, OR, IF, IR, GF, GR, EF, ER, PF, and PR are as follows:

[0021] OF: 5'-AAAGCTTGCCTTGAGTGCTT-3';

[0022] OR: 5'-TTAAGCAGTGGGTTCCCTTG-3';

[0023] IF: 5'-GCCCGTCTGTGTTAGGACTC-3';

[0024] IR: 5'-GGCTCGACCTGGTCTAACAA-3';

[0025] GF: 5'-AGGTGCACACAGCAAGAGG-3';

[0026] GR: 5'-CCTCCAATTCCCCTATCAT-3';

[0027] EF: 5'-CATATTGTGAGATTAATRRM-3';

[0028] ER: 5'-TGGAGCTGTTTAATGCCCCAGA-3';

[0029] PF: 5'-AACAGGGTTGTTGGAAATGC-3';

[0030] PR: 5'-TGATCCTTTCCATCCCTGTG-3'.

[0031] A third aspect of the present invention provides a method for screening and identifying unique recombinant strains and prevalent recombinant strains of human immunodeficiency virus type 1. The method involves first screening out suspected new subtypes of strains based on gene fragment sequences, then specifically amplifying the full-length sequences of these strains, further determining recombination breakpoints and establishing phylogenetic trees for comparison, and finally comprehensively judging whether they are new URFs or CRFs.

[0032] The specific steps are as follows:

[0033] Initial screening methods:

[0034] To prevent artificial recombination during the initial screening process due to segmented amplification, after reverse transcription, the first round of nested PCR amplification uses the full-length gene sequence, while the second round uses segment amplification for each gene fragment.

[0035] Because the HIV-1 content in the sample is extremely low, it is difficult to produce the required amount of product for sequencing with a single round of amplification. Therefore, two rounds of amplification are typically performed using nested PCR. The first round of amplification uses the outer primers OF and OR, which have the base sequences listed in the sequence listings SEQ ID NOs. 1 and 2. The second round of amplification builds on the first round, using inner primers from the gag, env, and pol gene regions for separate amplification and sequencing analysis. The inner primers GF, GR, EF, ER, PF, and PR from the gag, env, and pol gene regions have the base sequences listed in the sequence listings SEQ ID NOs. 5 to 10. Sequences from the three gene regions are analyzed for subtypes, and strains of known subtypes are eliminated. Mosaic maps of the remaining strains are created, and breakpoint locations are recorded. These maps are then compared with mosaic maps of publicly available CRFs to identify strains suspected of having new breakpoints, allowing for further full-length amplification and confirmation.

[0036] Confirmation of new URFs or CRFs strains:

[0037] Primers for amplifying the full-length gene sequence were used to amplify the full-length sequence of suspected new subtype strains. After splicing the full-length sequence, a mosaic map was created, recording breakpoint locations and subtypes, and a phylogenetic tree was constructed. This tree was compared with existing mosaic maps of CRFs to comprehensively determine whether it was a new URF. When a URF strain was screened and confirmed, if the number of the same URF strain was ≥3, it constituted an epidemic trend in the population, and was preliminarily identified as a CRF strain. The sequence was then uploaded to the HIV sequence database for final identification, naming, and publication. Given the low full-length amplification rate in previous literature, this invention employs a supplementary system. Without opening the container, trehalose is used to protect easily inactivated enzymes during the reaction process, and mineral oil is used to insulate the reaction system to prevent premature consumption of the supplementary system or enzyme inactivation.

[0038] This invention provides a low-cost and high-efficiency process and method for screening new unique recombinant viral strains (URFs) or prevalent recombinant viral strains (CRFs). It overcomes the risk of artificial recombination that often occurs when traditional full-length amplification involves fragmented amplification followed by splicing. Through the aforementioned technical improvements, this invention directly amplifies the full length and improves the efficiency and success rate of full-length amplification. The method provided by this invention is more accurate and reliable in screening and identifying new URFs or CRFs, providing fundamental technical support for monitoring HIV-1 strain recombination and evolution.

[0039] A fourth aspect of the present invention provides an application of the above-described primer set.

[0040] The primer set provided by this invention references the sequences of popular and recombinant HIV strains, and primers are designed in conserved regions to enable amplification of different HIV subtypes. This primer set is used for amplifying the full-length HIV-1 gene and amplifying mutant regions.

[0041] Nested PCR amplification was performed using two sets of primers when amplifying the full-length gene sequence.

[0042] First, the full-length sequence was reverse transcribed, and then the full-length sequence was amplified using nested PCR. The primers for the first round of nested PCR amplification were OF and OR, and the primers for the second round of nested PCR amplification were IF and IR.

[0043] Specifically, the steps include the following:

[0044] S1. Extract viral RNA from the sample;

[0045] S2. Perform full-length reverse transcription to cDNA. After a period of time, stop the reverse transcription. Without opening the cap, invert and mix the contents, then add the supplementary system from the inside of the cap into the reaction system to continue the reaction, in order to ensure the activity and fidelity of the enzyme during the long reaction.

[0046] S3. Using the above-mentioned outer primers OF and OR, the first round of nested PCR amplification of the full-length HIV-1 gene sequence was performed. After a period of time, the amplification was interrupted. Without opening the lid, the mixture was inverted and then the supplementary system on the lid was briefly flicked into the reaction system to continue the amplification reaction. This was done to overcome the decrease in enzyme fidelity and activity during the amplification of long fragments.

[0047] S4. Use the above-mentioned inner primers IF and IR to perform the second round of nested PCR amplification. After the amplification has been running for a period of time, interrupt it. Without opening the lid, invert and mix the mixture, then briefly flick the supplementary system from the inside of the lid into the reaction system to continue the amplification reaction. This is to overcome the decrease in enzyme fidelity and activity during long fragment amplification.

[0048] S5. The products from the second round of nested PCR amplification were subjected to electrophoresis. Samples with a 9kbp band at the target position were sequenced and the sequences were assembled for analysis.

[0049] Preferably, in step S1, viral RNA can be extracted from the sample using a commercially available automated extractor or a manual column extraction method.

[0050] Preferably, in step S2, the reverse transcription system for the full-length HIV-1 is as follows: This step uses a dual-system approach, including System 1 and System 2: System 1 is the standard reverse transcription reaction system, and System 2 is a supplementary system. System 1 (20 μL) contains 1.2 μL of dNTPs (10 mM), 2.0 μL of 10X buffer, 1 μL of Superscriptase III, 0.4 μL of RNase inhibitor, 0.8 μL of oligo-dT (50 mM) primers, and 14.6 μL of viral RNA template. System 2 contains 1 μL of Superscriptase III and 4 μL of 10% trehalose.

[0051] In this reaction tube, System 1 is added to the bottom, mineral oil is added above System 1 for insulation, and System 2 is added to the inside of the cap. System 1 in the reaction tube is reacted at 48°C for 90 minutes (without using a heated cap to prevent enzyme inactivation in System 2). Then, without opening the cap, System 2 is inverted and mixed, and then briefly transferred to the bottom of the reaction tube. The mixed system is then reacted again at 48°C for an additional 90 minutes.

[0052] The reason for supplementing System 2 to System 1 is that during reverse transcription of the full-length fragment, the fidelity and activity of the enzyme decrease during prolonged heating. By supplementing with System 2, the quantity and length of the reverse transcription product are ensured.

[0053] Preferably, in step S3, the first round of full-length PCR amplification uses a dual-system amplification system using reaction system 1 and supplementary system 2 to ensure enzyme activity and fidelity during prolonged heating. The Taq enzyme used is Hot Start LA Taq enzyme, which is specifically designed for amplifying long fragments. Reaction system 1 consists of: 2.5 μL of 10* buffer, 5 μL of MgCl2, 2.5 μL of dNTPs, 0.5 μL of 20 μM primer OF, 0.5 μL of 20 μM primer OR, 0.5 μL of Hot Start LA Taq enzyme, 8.5 μL of ddH2O, and 5 μL of the cDNA template reverse-transcribed in step S2. Supplementary system 2 consists of: 1 μL of Hot Start LA Taq enzyme and 4 μL of 10% trehalose.

[0054] Reaction system 1 was added to the bottom of the reaction tube, 20 μL of mineral oil was added above the reaction system 1 for heat insulation, and supplementary system 2 was added to the inner side of the cover of the reaction tube.

[0055] The first round of PCR amplification program was: 94℃ for 3 minutes; 94℃ for 15 seconds, 68℃ for 10 minutes, 20 cycles; after mixing by inverting, the supplementary system 2 in the cap was centrifuged into the reaction system 1 at the bottom of the reaction tube, and the amplification program was set to: 94℃ for 15 seconds, 68℃ for 10 minutes, 15 cycles.

[0056] Preferably, in step S4, the second round of nested PCR amplification uses both Reaction System 1 and Supplemental System 2 to ensure enzyme activity and fidelity during prolonged heating. The Taq enzyme used is Hot Start LA Taq, specifically designed for amplifying long fragments. Since the full-length second-round PCR product is used for 28 sequencing runs, a 100 μL reaction system 1 is prepared as follows: 10 μL 10* buffer, 20 μL MgCl₂, 10 μL dNTPs, 2 μL 20 μM primer IF, 2 μL 20 μM primer IR, 2 μL Hot Start LA Taq enzyme, and 34 μL ddH₂O. The first-round product from step S3 serves as a template (20 μL). Supplemental System 2 is prepared as follows: 10 μL Hot Start LA Taq enzyme and 8 μL 10% trehalose.

[0057] Reaction system 1 was added to the bottom of the reaction tube, 20 μL of mineral oil was added above the reaction system 1 for heat insulation, and supplementary system 2 was added to the inner side of the cover of the reaction tube.

[0058] The second round of PCR amplification program was: 94℃ for 3 minutes; 94℃ for 15 seconds, 68℃ for 10 minutes, 20 cycles; after removing and mixing the contents of the cap, the supplementary system 2 inside the cap was transferred to the reaction system 1 at the bottom of the reaction tube, and the amplification program was set to: 94℃ for 15 seconds, 68℃ for 10 minutes, 15 cycles.

[0059] Mosaic diagrams and phylogenetic trees are constructed based on the amplified and spliced ​​full-length sequences. The breakpoints and subtypes in the mosaic diagrams, as well as the branches and phylogenetic relationships in the phylogenetic trees, are used to comprehensively determine whether a new URFs or CRFs strain is present.

[0060] Preferably, the sequencing primers in step S5 are conventional full-length sequencing primers, as shown in List 1. Due to the limitations of first-generation sequencing technology, the effective sequencing length of a single reaction is generally around 900 bp or shorter. Therefore, a stacked sequencing approach is used to sequence multiple fragments and then assemble them. The sequencing primers are shown in Table 1 and are common, conventional sequencing reactions. The sequencing results are assembled using the SeqMan program in Lasergene software. The assembled sequences are exported in Fasta format and subjected to recombination analysis to identify breakpoint locations, draw a mosaic diagram, construct a phylogenetic tree, and comprehensively determine whether the amplified sequences are new URFs or CRFs strains.

[0061] Table 1 Conventional sequencing primers used for sequencing

[0062]

[0063]

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] (1) The method for screening and identifying unique recombinant strains and circulating recombinant strains of human immunodeficiency virus type 1 in this invention first performs subtype screening and compares the mosaic patterns of different gene regions in a large number of samples, eliminates strains with known subtypes and known mosaic patterns, then performs full-length amplification on some suspected new subtype strains to lock and reduce the target range, and then performs full-length amplification on the target strains and compares them; this method and process not only provides ideas for the screening and discovery of new subtypes, but also reduces economic and time costs and improves the efficiency of new subtype screening;

[0066] (2) Most literature currently identifies new URFs or CRFs, but only performs final discrimination without detailing screening methods from a large number of samples. Alternatively, it may involve amplifying the full-length sequence of all samples before screening, which is time-consuming, labor-intensive, and costly in terms of amplification and sequencing. Furthermore, some literature reports a full-length amplification rate of only 66%. Traditional full-length amplification, with its segmented amplification, carries the risk of artificial recombination, and the resulting full-length sequence has a certain probability of originating from different strains within the quasi-species. This invention employs a first round of full-length amplification, followed by a second round amplifying only regions prone to breakpoints and mutations. Preliminary screening is performed based on breakpoints and subtypes before full-length amplification to prevent artificial recombination and ensure that the amplified genes are fragments from the same strain. In addition, the reason why traditional methods of segmented amplification are used is that direct amplification of the full length takes a long time, and Taq enzyme is prone to inactivation or low efficiency, which reduces the success rate of long fragment amplification. In this invention, the method of adding a supplementary system is used in the amplification technology, which improves the success rate of amplifying the full length. The addition of trehalose in the supplementary system effectively protects the enzyme, ensuring the enzyme's activity and fidelity. Furthermore, the cap is not opened during the amplification process to prevent contamination, resulting in higher sensitivity and amplification success rate.

[0067] (3) The method for screening and identifying unique recombinant strains and popular recombinant strains of human immunodeficiency virus type 1 provided by the present invention has the characteristics of low cost and high efficiency. Instead of performing full-length amplification on all candidate samples in the traditional way, it is changed to performing amplification analysis and screening on easily mutated regions, which narrows the target range that needs to be amplified to the full length, and performs amplification and analysis more specifically. It overcomes the risk of artificial recombination caused by multi-fragment full-length amplification. Through technical improvements, the amplification success rate is increased. The entire screening and identification process is clear and detailed, and has more application value and strong practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0069] Figure 1 For large-sample screening, the mosaic plots of gag, env, and pol for the same sample, as well as the breakpoints and subtype characteristics (showing some samples);

[0070] Figure 2 Mosaic diagrams of the full-length genomes of several suspected new URFs or CRFs, as well as breakpoint and subtype analysis diagrams (showing some samples);

[0071] Figure 3 A whole-genome phylogenetic tree was constructed for several sample strains suspected to be new URFs or CRFs and for the identified reference strains. DETAILED DESCRIPTION

[0072] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0073] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0074] Unless otherwise specified, all reagents and raw materials used in this invention are commercially available products or products that can be prepared by known methods.

[0075] Example 1

[0076] This embodiment provides a method and procedure for preliminary screening of suspected new subtype strains based on gene fragment sequences.

[0077] The initial screening uses reverse transcription of the full-length sequence, the first round of PCR amplifies the full-length sequence, and the second round of PCR amplifies some gene sequences that are prone to breakpoints and mutations.

[0078] The first round of nested PCR amplification primers were OF and OR. The second round of nested PCR amplified gene fragments in the gag, env, and pol genes that are prone to breakpoints and mutations, using GF and GR, EF and ER, and PF and PR primers, respectively.

[0079] The nucleotide sequence of OF is shown in SEQ ID NO.1;

[0080] The nucleotide sequence of OR is shown in SEQ ID NO.2;

[0081] The nucleotide sequence of GF is shown in SEQ ID NO.5;

[0082] The nucleotide sequence of GR is shown in SEQ ID NO.6;

[0083] The nucleotide sequence of EF is shown in SEQ ID NO.7;

[0084] The nucleotide sequence of ER is shown in SEQ ID NO.8;

[0085] The nucleotide sequence of PF is shown in SEQ ID NO.9;

[0086] The nucleotide sequence of PR is shown in SEQ ID NO.10;

[0087] Specifically, the nucleotide sequences of OF, OR, GF, GR, EF, ER, PF, and PR are as follows:

[0088] OF: 5'-AAAGCTTGCCTTGAGTGCTT-3';

[0089] OR: 5'-TTAAGCAGTGGGTTCCCTTG-3';

[0090] GF: 5'-AGGTGCACACAGCAAGAGG-3';

[0091] GR: 5'-CCTCCAATTCCCCTATCAT-3';

[0092] EF: 5'-CATATTGTGAGATTAATRRM-3';

[0093] ER: 5'-TGGAGCTGTTTAATGCCCCAGA-3';

[0094] PF: 5'-AACAGGGTTGTTGGAAATGC-3';

[0095] PR: 5'-TGATCCTTTCCATCCCTGTG-3';

[0096] The primer set provided in this embodiment references various prevalent HIV strains in the HIV database. Multiple sequence alignment was performed on the HIV subtype sequences to identify conserved regions for different subtypes. Primers designed within these conserved regions can amplify different HIV subtypes. Given the high variability of HIV-1, this invention introduces degenerate bases (mixed bases) to ensure good binding to the template even when base mutations occur in the primer regions for different subtypes. In primers EF, M stands for A / C, and R stands for A / G.

[0097] This embodiment also provides an efficient and more accurate method and process for screening suspected new URFs or CRFs, including the following steps:

[0098] S1. Extract viral RNA from the sample;

[0099] S2. Perform reverse transcription to cDNA. After a period of time, the reverse transcription is interrupted. Without opening the cap, invert and mix the contents, then add the supplementary system from the inside of the cap into the reaction system to continue the reaction, in order to ensure the activity and fidelity of the enzyme during the long reaction.

[0100] S3. Use the above-mentioned outer primers OF and OR to perform the first round of PCR on the full-length HIV-1 gene sequence. After a period of amplification, interrupt the process. Without opening the lid, invert and mix the mixture, then add the supplementary system from the inside of the lid into the reaction system to continue amplification. This is to overcome the decrease in enzyme fidelity and activity caused by the long amplification time when amplifying long fragments.

[0101] S4. Use the above-mentioned inner primers GF and GR, EF and ER, PF and PR to amplify the gene fragments gag, env, and pol in the HIV-1 gene that are prone to breakpoints and mutations.

[0102] S5. Perform electrophoresis on the second round of nested PCR amplification products. Sequencing is performed on samples at the target band position, and the sequences are assembled for analysis.

[0103] Preferably, in step S1, viral RNA can be extracted from the sample using a commercially available automated extractor or a manual column extraction method.

[0104] Preferably, in step S2, the reverse transcription system for the full-length HIV-1 is as follows: This step has two systems: System 1 is the reverse transcription system, and System 2 is a supplementary system. System 1 is 20 μL, containing 1.2 μL of dNTPs (10 mM), 2.0 μL of 10X buffer, 1 μL of Superscriptase III, 0.4 μL of RNase inhibitor, 0.8 μL of oligo-dT (50 mM) primers, and 14.6 μL of viral RNA template. The blank control uses an equal volume of deionized water instead of template. System 2 is 5 μL, containing 1 μL of Superscriptase III and 4 μL of 10% trehalose.

[0105] System 1 was added to the bottom of the reaction tube, and mineral oil was used to insulate between System 1 and System 2. System 2 was added to the inside of the tube cap. After System 1 was reacted at 48°C for 90 minutes (the hot cap was not used to prevent enzyme inactivation in System 2), the cap was not opened. The mixture was then inverted and briefly transferred to the bottom of the tube. The mixed system was then reacted again at 48°C for an additional 90 minutes. The reason for using System 2 to supplement System 1 is that during reverse transcription of the full-length fragment, the enzyme fidelity and activity decrease during prolonged heating. Supplementing with System 2 ensures the quantity and length of the reverse transcription product.

[0106] Preferably, in step S3, the first round of full-length PCR amplification still uses a dual-system amplification with reaction system 1 and supplementary system 2 to ensure enzyme activity and fidelity during prolonged heating. The Taq enzyme used is Hot Start LA Taq enzyme specifically designed for amplifying long fragments. Reaction system 1 (25 μL) consists of: 2.5 μL 10*buffer, 5 μL MgCl2, 2.5 μL dNTPs, 0.5 μL 20 μM primer OF, 0.5 μL 20 μM primer OR, 0.5 μL Hot Start LA Taq enzyme, 8.5 μL ddH2O, and 5 μL cDNA template from step S2. The blank control uses an equal volume of deionized water instead of template. Supplementary system 2 (5 μL) consists of 1 μL Hot Start LA Taq enzyme and 4 μL 10% trehalose.

[0107] Reaction system 1 was added to the bottom of the reaction tube, 20 μL of mineral oil was added above the reaction system 1 for heat insulation, and supplementary system 2 was added to the inner side of the cover of the reaction tube.

[0108] The first round of PCR amplification program is: 94℃ for 3 minutes; 94℃ for 15 seconds, 68℃ for 10 minutes, and 20 cycles; choose not to use the hot cover, take out and invert to mix, then centrifuge the supplementary system 2 in the cover into the reaction system 1 at the bottom of the reaction tube, and set the amplification program to: 94℃ for 15 seconds, 68℃ for 10 minutes, and 15 cycles.

[0109] Preferably, in step S4, the system for performing nested PCR second round amplification of the gene fragments gag, env, and pol, which are prone to breakpoints and mutations in the HIV-1 gene, using the inner primers GF and GR, EF and ER, and PF and PR is as follows: the template used in the second round of amplification is the amplified product of the first round, and the reagent used is PremixTaq TM The amplification products from this step are used for sequencing. The total reaction volume 1 (50 μL) consists of 25 μL Premix Taq, 1 μL of 20 μM upstream inner primer, 1 μL of 20 μM downstream inner primer, 18 μL of deionized water, and 5 μL of template (first-round amplification product). When amplifying *gag*, the amplification and sequencing primers are GF and GR; when amplifying *env*, the amplification and sequencing primers are EF and ER; and when amplifying *pol*, the amplification and sequencing primers are PF and PR. An equal volume of deionized water is used as the blank control instead of the template.

[0110] The second round of nested PCR amplification program was as follows: 94℃ for 3 minutes; 94℃ for 5 seconds, 55℃ for 5 seconds, 72℃ for 1 minute, 30 cycles; the hot cap temperature was 105℃.

[0111] The sequencing results were assembled using the SeqMan program in Lasergene software, and the assembled sequences were exported in Fasta format. 321 strains successfully amplified and sequenced all three gene regions (gag, env, and pol). After removing sequences from known subtypes, 110 strains remained. The remaining sequences were then analyzed to determine if the amplified sequences were suspected to be new URFs or CRFs. Homologous sequences with the same mosaic map as known CRFs or branches at the same end in the phylogenetic tree were removed, retaining strains with new mosaic maps and different phylogenetic branches, ultimately resulting in 42 strains. See [link to relevant documentation] Figure 1 Mosaic images of the same sample (env, gag, pol). In the original mosaic image, different colors represented different subtypes; after being converted to black and white, they are distinguished by different shades of gray. According to... Figure 1 Mosaic maps of gene regions prone to mutation were compared with those of published CRFs, and no identical or similar maps were found, suggesting a possible new breakpoint method. Due to space limitations, [further details omitted]. Figure 1 Some of the suspected new URFs strains that were screened out are shown.

[0112] Example 2

[0113] This embodiment provides a method and procedure for amplifying the full genome sequence of candidate strains, as well as a method and procedure for identifying URFs or CRFs.

[0114] In this embodiment, the full-length sequence was first reverse transcribed, and then amplified using nested PCR. The primers for the first round of nested PCR amplification were OF and OR, and the primers for the second round of nested PCR amplification were IF and IR.

[0115] S1. Extract viral RNA from the sample (same as in Example 1);

[0116] S2. Perform full-length reverse transcription to cDNA. After the reverse transcription has been running for a period of time, stop it. Without opening the cap, invert and mix the contents, then add the supplementary system from the inside of the cap into the reaction system to continue the reaction, in order to ensure the activity and fidelity of the enzyme during the long reaction (same as in Example 1).

[0117] S3. Using the above-mentioned outer primers OF and OR, the first round of nested PCR amplification of the full-length HIV-1 gene sequence was performed. The amplification was interrupted after a period of time. Without opening the lid, the mixture was inverted and then the supplementary system on the lid was thrown into the reaction system to continue the amplification reaction. This was used to overcome the decrease in enzyme fidelity and activity during long fragment amplification (same as Example 1).

[0118] S4. Perform the conventional second round of nested PCR using the above-mentioned inner primers IF and IR. After a period of time, interrupt the amplification process. Without opening the lid, invert and mix the mixture, then flick the supplementary system from the lid into the reaction system to continue the amplification reaction. This is to overcome the decrease in enzyme fidelity and activity during long fragment amplification.

[0119] S5. The products from the second round of nested PCR amplification were subjected to electrophoresis. Samples with a 9kbp band at the target position were sequenced and the sequences were assembled for analysis.

[0120] Preferably, in step S1, viral RNA can be extracted from the sample using a commercially available automated extractor or a manual column extraction method.

[0121] Preferably, in step S2, the reverse transcription system for the full-length HIV-1 is as follows: This step uses a dual-system approach, including System 1 and System 2: System 1 is the standard reverse transcription reaction system, and System 2 is a supplementary system. System 1 consists of 1.2 μL of dNTPs (10 mM), 2.0 μL of 10X buffer, 1 μL of Superscriptase III, 0.4 μL of RNase inhibitor, 0.8 μL of oligo-dT (50 mM) primer, and 14.6 μL of viral RNA template. System 2 consists of 1 μL of Superscriptase III and 4 μL of 10% trehalose.

[0122] In this reaction tube, System 1 is added to the bottom, mineral oil is added above System 1 for insulation, and System 2 is added to the inside of the cap. System 1 in the reaction tube is reacted at 48°C for 90 minutes (without using a heated cap to prevent enzyme inactivation in System 2). Then, without opening the cap, System 2 is mixed by inverting the tube and briefly poured to the bottom. The mixed system is then reacted again at 48°C for another 90 minutes.

[0123] The reason for supplementing System 2 to System 1 is that during reverse transcription of the full-length fragment, the fidelity and activity of the enzyme decrease during prolonged heating. By supplementing with System 2, the quantity and length of the reverse transcription product are ensured.

[0124] Preferably, in step S3, the first round of full-length PCR amplification uses a dual-system amplification system using reaction system 1 and supplementary system 2 to ensure enzyme activity and fidelity during prolonged heating. The Taq enzyme used is Hot Start LA Taq enzyme, which is specifically designed for amplifying long fragments. Reaction system 1 consists of: 2.5 μL of 10* buffer, 5 μL of MgCl2, 2.5 μL of dNTPs, 0.5 μL of 20 μM primer OF, 0.5 μL of 20 μM primer OR, 0.5 μL of Hot Start LA Taq enzyme, 8.5 μL of ddH2O, and 5 μL of the cDNA template reverse-transcribed in step S2. Supplementary system 2 consists of: 1 μL of Hot Start LA Taq enzyme and 4 μL of 10% trehalose.

[0125] Reaction system 1 was added to the bottom of the reaction tube, 20 μL of mineral oil was added above the reaction system 1 for heat insulation, and supplementary system 2 was added to the inner side of the cover of the reaction tube.

[0126] The first round of PCR amplification program is: 94℃ for 3 minutes; 94℃ for 15 seconds, 68℃ for 10 minutes, and 20 cycles; choose not to use the hot cover, take out and invert to mix, then centrifuge the supplementary system 2 in the cover into the reaction system 1 at the bottom of the reaction tube, and set the amplification program to: 94℃ for 15 seconds, 68℃ for 10 minutes, and 15 cycles.

[0127] Preferably, in step S4, the second round of nested PCR amplification uses both Reaction System 1 and Supplemental System 2 to ensure enzyme activity and fidelity during prolonged heating. The Taq enzyme used is Hot Start LA Taq, specifically designed for amplifying long fragments. Since the full-length second-round PCR product is used for 28 sequencing runs, a 100 μL reaction system 1 is prepared as follows: 10 μL 10* buffer, 20 μL MgCl₂, 10 μL dNTPs, 2 μL 20 μM primer IF, 2 μL 20 μM primer IR, 2 μL Hot Start LA Taq enzyme, and 34 μL ddH₂O. The first-round product from step S3 serves as a template (20 μL). Supplemental System 2 is prepared as follows: 10 μL Hot Start LA Taq enzyme and 8 μL 10% trehalose.

[0128] Reaction system 1 was added to the bottom of the reaction tube, 20 μL of mineral oil was added above the reaction system 1 for heat insulation, and supplementary system 2 was added to the inner side of the cover of the reaction tube.

[0129] The second round of PCR amplification program is: 94℃ for 3 minutes; 94℃ for 15 seconds, 68℃ for 10 minutes, and 20 cycles; choose not to use the hot cover, take it out and invert to mix, then centrifuge the supplementary system 2 in the cover into the reaction system 1 at the bottom of the reaction tube, and set the amplification program to: 94℃ for 15 seconds, 68℃ for 10 minutes, and 15 cycles.

[0130] Forty-two suspected novel subtypes of the virus strains screened in Example 1 were amplified to obtain 35 full-length gene sequences. Using full-length sequencing primers (see Table 1), each strain was sequenced 28 times, and the sequences were then assembled. Mosaic diagrams and phylogenetic trees were constructed based on the assembled full-length sequences. The breakpoints in the mosaic diagrams, subtypes, and phylogenetic tree branches and phylogenetic relationships were used to comprehensively determine whether the strain was a new URFs or CRFs strain.

[0131] The results of the breakpoint and subtype analysis are shown in Table 2. ～ 5, which shows the breakpoint start and end positions of the four amplified full-genome sequences (due to space, only some strain results are shown), as well as the subtypes of the fragments. After comparison with the 132 published CRFs, they are new URFs; Figure 2 The full-length mosaic diagram of the four strains was shown. After comparison with the 132 published cases, they were identified as new URFs. The phylogenetic tree was established by combining the screened URFs with the reference strains (only 9 of them were shown, see Figure 3 YN211 and CRF86_BC share homology at the end of the same branch, thus excluding them as new URFs. In the mosaic image, different colors originally represented different subtypes; after being converted to a black and white image, they are distinguished by different shades of gray. The remaining 8 cases starting with YN in the phylogenetic tree form separate evolutionary branches and show no homology with the reference strain, thus being identified as new URFs. When the number of each new URF reaches 3 or more, they are uploaded to the HIV circulating recombinant forms (CRFs) website for final evaluation and naming. In this example, YN242 / YN306 and YN381 / YN417 show homology (see...). Figure 3 Each group contains 2 cases. When a new homologous strain is found, it can be uploaded to the website for final evaluation, confirmation, and naming of the new CRFs.

[0132] Table 2 Breakpoint and Subtype Analysis of YN242

[0133]

[0134]

[0135] Table 3. Breakpoint and Subtype Analysis of YN205

[0136]

[0137] Table 4 YN211 breakpoint and subtype analysis

[0138]

[0139]

[0140] Table 5 YN233 breakpoint and subtype analysis

[0141]

[0142] Comparative Example 1

[0143] Comparison of full-length amplification results for samples with different viral loads, under conditions of no supplementation and with supplementation: (for samples with viral load <10...) 3 copies / mL, 10 3 -10 5 copies / mL, >10 6 Ten samples within the range of 10 copies / mL were selected to compare the two methods.

[0144] The method using the supplementary system employs the amplification method and process described in Example 2 above.

[0145] The standard amplification method without supplementary systems is as follows:

[0146] Reverse transcription was performed first. The 30 μL reverse transcription system consisted of: 1.8 μL dNTPs, 1.2 μL oligo-dT, 6.0 μL 5X buffer, 2.4 μL 0.1M DTT, 1.2 μL reverse transcriptase, 0.6 μL RNase inhibitor, 1.8 μL ddH2O, and 15.0 μL RNA. The reaction was carried out at 45°C for 1.5 hours.

[0147] The first round amplification reaction system of nested PCR was as follows: 2×LA Mixture 25 μL, 1 μL of 10 pmol / μL upstream outer primer, 1 μL of 10 pmol / uL downstream outer primer, 5 μL of reverse transcribed cDNA, and 18 μL of deionized water.

[0148] The second round of nested PCR amplification reaction system was as follows: 25 μL of 2×LA Mixture, 1 μL of 10 pmol / μL upstream inner primer, 1 μL of 10 pmol / μL downstream inner primer, 5 μL of reverse transcribed cDNA, and 18 μL of deionized water.

[0149] The reaction conditions for both rounds of nested PCR were: 94°C for 2 minutes; 94°C for 10 seconds, 68°C for 9 minutes, 30 cycles; and the heated lid temperature was 105°C.

[0150] The test results are shown in Table 6.

[0151] Table 6 Comparative experimental results of full-length amplification by conventional method and dual system method

[0152] <![CDATA[<10 3 copies / mL]]> <![CDATA[10 3 -10 6 copies / mL]]> <![CDATA[>10 6 copies / mL]]> Conventional method 1 / 10 3 / 10 6 / 10 Dual-system method 3 / 10 7 / 10 9 / 10

[0153] The experimental results in Table 6 show that the dual-system method of the present invention has a higher amplification success rate than the conventional method in different viral load ranges; especially in the case of <10 3 copies / mL, 10 3 -10 6 In the two ranges of 100 copies / mL and 100 copies / mL, the use of the supplementation system of the present invention not only improves the success rate of amplification but also significantly improves the detection sensitivity. This shows that the amplification method of the amplification primers and supplementation system of the present invention has higher sensitivity and amplification efficiency than conventional methods in experiments with low and medium viral loads.

[0154] In summary, this invention employs a first round of full-length amplification, followed by a second round amplifying only regions prone to breakpoints and mutations. Initial screening is performed based on breakpoints and subtypes before full-length amplification is performed on suspected new subtype strains, preventing artificial recombination and ensuring that the amplified genes are fragments from the same strain. Furthermore, the reason for segmented amplification in traditional methods is that direct full-length amplification is time-consuming, easily leading to Taq enzyme inactivation or inefficiency, thus reducing the success rate of long fragment amplification. This invention uses a supplementary system to improve the success rate of full-length amplification. The addition of trehalose to the supplementary system effectively protects the enzyme, ensuring its activity and fidelity. Moreover, the amplification process is conducted without opening the cap to prevent contamination, resulting in higher sensitivity and amplification success rate.

[0155] The method for screening and identifying unique recombinant strains and circulating recombinant strains of human immunodeficiency virus type 1 provided by this invention is characterized by low cost and high efficiency. Compared with the traditional method of full-length amplification and sequencing of each sample, this method changes to amplification analysis and screening of easily mutated regions, which improves screening efficiency. Through initial screening, suspected new subtypes can be screened from a large number of candidate samples, narrowing the target range of full-length amplification and allowing for more targeted amplification and analysis. The entire screening process is clear and detailed, has greater application value, and has stronger practical application value.

[0156] The above examples are used to illustrate the present invention, which are only used to help understand the present invention and are not intended to limit the present invention. Those skilled in the art can make several simple deductions, modifications or substitutions based on the concept of the present invention.

Claims

1. A method for screening and identifying unique recombinant strains and prevalent recombinant strains of human immunodeficiency virus type 1, characterized in that: First, the amplified gag, env, and pol gene sequences, which are prone to breakpoint mutations and are used to amplify the breakpoint mutation-prone regions of HIV-1, were subjected to genotyping analysis. After filtering out known genotypes, the remaining sample sequences suspected of novel subtypes were identified using jpHMM for breakpoint location and mosaic mapping. A secondary screening was performed, in which samples with identical mosaic maps to known CRFs were removed, retaining samples suspected of novel subtypes. When a sample suspected of a new subtype is screened out, the full-length gene sequence is amplified, the breakpoint position of the full-length gene sequence is identified using jpHMM, and a mosaic diagram is drawn. An evolutionary tree is established together with the reference sequence, and a comprehensive judgment is made based on the mosaic diagram and evolutionary tree results to determine whether it is a new URFs strain.

2. The method for screening and identifying unique recombinant strains and prevalent recombinant strains of human immunodeficiency virus type 1 according to claim 1, characterized in that: Primers for amplifying the full-length gene sequence of HIV-1, including an outer primer pair for the first round of nested PCR amplification of the full-length gene sequence and an inner primer pair for the second round of nested PCR amplification of the full-length gene sequence; The outer primer pair used for the first round of full-length gene sequence amplification was OF and OR, and the inner primer pair used for the second round of full-length gene sequence amplification was IF and IR; The nucleotide sequence of OF is shown in SEQ ID NO.1; The nucleotide sequence of OR is shown in SEQ ID NO. 2; The nucleotide sequence of IF is shown in SEQ ID NO. 3; The nucleotide sequence of IR is shown in SEQ ID NO.

4.

3. The method for screening and identifying unique recombinant strains and prevalent recombinant strains of human immunodeficiency virus type 1 according to claim 2, characterized in that: The primers for amplifying fragments of the gag, env, and pol gene sequences in the HIV-1 breakpoint mutation-prone region include a primer pair for the first round of nested PCR amplification and a primer pair for the second round of amplification; the primers for the first round of nested PCR amplification of the three gene fragment regions all use outer primers OF and OR for amplifying the full-length gene sequence; in the primer pair for the second round of nested PCR amplification, the amplification primers for the gag, env, and pol gene fragments correspond to GF, GR, EF, ER, PF, and PR, respectively; The nucleotide sequence of GF is shown in SEQ ID NO.5; The nucleotide sequence of GR is shown in SEQ ID NO.6; The nucleotide sequence of EF is shown in SEQ ID NO.7; The nucleotide sequence of ER is shown in SEQ ID NO.8; The nucleotide sequence of PF is shown in SEQ ID NO.9; The nucleotide sequence of PR is shown in SEQ ID NO.

10.

4. The method for screening and identifying unique recombinant strains and prevalent recombinant strains of human immunodeficiency virus type 1 according to claim 1, characterized in that: When screening and confirming as URFs strains, when the number of the same URFs strain is ≥3, it constitutes an epidemic trend in the population, and it is preliminarily determined to be a CRFs strain, and the sequence is uploaded to the HIV sequence database for final determination, naming and disclosure.

5. The method for screening and identifying unique recombinant strains and prevalent recombinant strains of human immunodeficiency virus type 1 according to claim 2, characterized in that: The invention also includes a method for amplifying a full-length gene sequence that is efficient and prevents artificial recombination. Some steps are amplified using a dual system, specifically including the following steps: S1. Extract viral RNA from samples; S2. Perform full-length reverse transcription into cDNA. After a period of time, interrupt the reverse transcription. Without opening the lid, invert and mix thoroughly, then throw the supplementary system inside the lid into the reaction system to continue the reaction to ensure the activity and fidelity of the enzyme during the long reaction. S3. Perform the first round of nested PCR amplification of the full-length HIV-1 gene sequence using the outer primers OF and OR. After a period of amplification, interrupt the amplification. Without opening the lid, invert and mix thoroughly, then throw the supplementary system inside the lid into the reaction system to continue the amplification reaction. This is to overcome the reduction in enzyme fidelity and activity during long fragment amplification. S4. Perform the second round of nested PCR amplification using inner primers IF and IR. After a period of amplification, interrupt the amplification. Without opening the lid, invert and mix thoroughly, then throw the supplementary system inside the lid into the reaction system to continue the amplification reaction. This is used to overcome the reduction in enzyme fidelity and activity during long fragment amplification. S5. The products of the second round of nested PCR were subjected to electrophoresis. The samples with the target band position of 9 kbp were sequenced and the sequences were spliced for analysis.

6. The method for screening and identifying unique recombinant strains and prevalent recombinant strains of human immunodeficiency virus type 1 according to claim 5, characterized in that: The dual system in step S2 includes system 1 and system 2: system 1 is a conventional reverse transcription reaction system, and system 2 is a supplementary system; system 2 includes 1 μL of Superscriptase III and 4 μL of 10% trehalose; wherein system 1 is added to the bottom of the reaction tube, mineral oil is added above system 1 for heat insulation, and system 2 is added to the inside of the lid of the reaction tube.

7. The method for screening and identifying unique recombinant strains and prevalent recombinant strains of human immunodeficiency virus type 1 according to claim 5, characterized in that: In step S2, system 1 in the reaction tube is reacted at 48°C for 90 minutes without using a heated lid to prevent inactivation of the enzyme in system 2. Then, without opening the lid, the tube is inverted and mixed, and system 2 on the inner side of the lid is instantly transferred to the bottom of the reaction tube. The mixed system is then reacted at 48°C for an additional 90 minutes.

8. The method for screening and identifying unique recombinant strains and prevalent recombinant strains of human immunodeficiency virus type 1 according to claim 5, characterized in that: In steps S3 and S4, both the first round of PCR amplification and the second round of PCR amplification are performed using dual-system amplification using reaction system 1 and supplementary system 2; in step S3, supplementary system 2 includes 1 μL of HotStart LA Taq enzyme and 4 μL of 10% trehalose; in step S4, supplementary system 2 includes 2 μL of HotStart LA Taq enzyme and 8 μL of 10% trehalose; wherein, reaction system 1 is added to the bottom of the reaction tube, mineral oil is added above reaction system 1 for heat insulation, and supplementary system 2 is added to the inner side of the lid of the reaction tube.

9. The method for screening and identifying unique recombinant strains and prevalent recombinant strains of human immunodeficiency virus type 1 according to claim 5, characterized in that: In step S3, the first round of PCR amplification program is: 94°C for 3 minutes; 94°C for 15 seconds, 68°C for 10 minutes, and 20 cycles; if the heated lid is not used, the tube is removed and mixed by inversion, and the supplementary system 2 in the lid is quickly transferred to the reaction system 1 at the bottom of the reaction tube. The amplification program is set to: 94°C for 15 seconds, 68°C for 10 minutes, and 15 cycles; In step S4, the second round of PCR amplification program is: 94°C for 3 minutes; 94°C for 15 seconds, 68°C for 10 minutes, 20 cycles; Choose not to use the hot cover, take out and invert to mix, and quickly transfer the supplementary system 2 in the cover to the reaction system 1 at the bottom of the reaction tube. Set the amplification program as follows: 94℃ for 15 seconds, 68℃ for 10 minutes, and 15 cycles.

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