A hepatitis c virus new subtype 6xp amplification primer and a drug-resistant mutation detection primer set

Abstract

This invention discloses a primer set for amplifying a novel HCV subtype 6xp and a primer set for detecting drug resistance mutations. The primer set for amplifying the novel HCV subtype 6xp includes nested PCR primers for amplifying the full length of the novel HCV subtype 6xp, while the primer set for detecting drug resistance mutations is a set of primers targeting drug resistance mutation sites in the three functional regions of NS3, NS5A, and NS5B. The primer set provided by this invention has high specificity and high amplification efficiency, enabling specific amplification of the entire genome of the novel HCV subtype 6xp and accurate detection of drug resistance mutation sites in the three functional regions. It is suitable for clinical diagnosis, antiviral treatment guidance, epidemiological surveys, and viral evolution research of this subtype, and has significant clinical application value and scientific research significance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and molecular in vitro diagnostics, specifically relating to a new HCV subtype 6xp amplification primer and a set of primers for detecting drug resistance mutations. Background Technology

[0002] Hepatitis C virus (HCV) is one of the main pathogens causing chronic liver disease. Its genome is highly variable, leading to a wide variety of genotypes and subtypes. Currently, HCV is classified into 8 major genotypes and 94 subtypes, with type 6 being the most complex and diverse. This genotypic diversity of HCV presents a series of challenges to clinical diagnosis, precise treatment planning, vaccine development, and epidemiological monitoring and control, thus requiring close monitoring and in-depth research.

[0003] Currently, the core treatment regimen for HCV in clinical practice involves pangenotypic direct antiviral agents (DAAs). These drugs exert their antiviral effects by specifically blocking key proteins or enzymes in the HCV life cycle. They mainly include NS3 / 4A protease inhibitors (PIs), NS5A inhibitors, NS5B nucleotide analogue inhibitors, and NS5B non-nucleotide analogue inhibitors. However, different genotypes and subtypes exhibit significant differences in sensitivity to DAAs, and with the widespread clinical use of DAAs, HCV drug resistance is becoming increasingly prominent. Current technology has confirmed that key drug resistance mutation sites in HCV are distributed across three different functional regions: NS3 / 4A, NS5A, and NS5B. Therefore, early identification of specific HCV subtypes and their drug resistance mutations in these three functional regions is a crucial prerequisite for developing precise adjuvant therapy strategies and improving treatment success rates.

[0004] In recent years, the applicant has discovered a novel HCV 6 subtype (named 6xp subtype in this invention) during epidemiological surveillance. This new subtype exhibits a genetic distance greater than 15% from other known HCV 6 subtypes and forms an independent phylogenetic cluster, clearly identifying it as a novel HCV 6 subtype not previously reported in existing technologies. However, existing technologies face the following technical bottlenecks in molecular diagnostics and drug resistance assessment for this newly emerging 6xp subtype: First, there is a lack of specific amplification tools for the entire genome of this new subtype. Existing universal full-length amplification primers have extremely low amplification efficiency or fail to amplify this new subtype, resulting in the inability to obtain its complete genomic information, and consequently, failing to provide a basis for subsequent subtype identification and drug resistance site detection. Second, existing drug resistance mutation detection primers cannot simultaneously cover the drug resistance sites scattered across the NS3, NS5A, and NS5B regions of this new subtype, easily leading to missing amplification of the target fragment, missed detection, or false detection, thus failing to meet the clinical needs for assessing the drug resistance of this new subtype. Therefore, there is an urgent need in this field to develop a highly efficient full-length amplification primer set and a drug resistance mutation detection primer set specifically for the novel HCV subtype 6xp, in order to fill the technological gap in molecular diagnosis and drug resistance assessment for this subtype. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a full-length amplification primer set for the novel HCV subtype 6xp, solving the technical problem that existing universal primers cannot efficiently amplify the entire genome of this novel subtype and cannot obtain complete genomic information. This invention also provides a detection primer set for drug resistance mutations in the novel HCV subtype 6xp, addressing the technical deficiencies of existing drug resistance detection primers that cannot simultaneously detect drug resistance sites in the multifunctional regions of this novel subtype and are prone to false negatives and false positives, thus providing technical support for the molecular diagnosis, drug resistance assessment, and clinical treatment of this novel subtype.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: 1. The primer set for the full-length amplification of the novel HCV subtype 6xp in this invention includes eight pairs of nested PCR amplification primers 6xp1F1 / R1 to 6xp8F2 / R2, with the following nucleotide sequences: 6xp1: F1 GCGACACTCCACCATGATC; R1 TMGCCCARTTAGCCAYCATGC; F2 CCATGATCACTCCCCTGTGAG; R2 ATGCTGTAATATAGCACGCCAAG; 6xp2: F1 TGTGGTGGTGTGTTCCTGGTTG; R1 TTCTCYAGAGCCGCCTCAGC; F2 GACAACTTTTCACCTTCCGG; R2 CCTCAGCCTGAGATATAAGCATCA; 6xp3: F1 TGCTCGTTTACCACTATGCCG; R1 GTGACGATMGTCCCCATMAGG; F2 TTGTCRACCGGYTTAATACATCTAC; R2 TGTTGGTGGTACGCCGTAATG; 6xp4: F1 TGACCTTGCCGTAGCGGTTG; R1 TCAAGGCTRAARTCRACCAYTTG; F2 CGATGGAGAAGAARGTCATCAC; R2 GTCACTGCCACRTTRCARTCAA; 6xp5: F1 GGTAGGCACTTGATCTTCTGTCAC; R1 CTGTTCATCCACTGGTTSGC; F2 AAGAARTGYGACGAGCTGGC; R2 CAGTARGTTGACCATGTCCTCC; 6xp6: F1 ACCCTCCTCCTGAACATCTTAG; R1 CACTTCCTGCCTCCATAGCA; F2 GCWTCTCAGCTTGCWCCAC; R2 TTCGCCTCAAGCAACTCAGC; 6xp7: F1 CTTACCAGTTACGCCATAGGG; R1 TGGTGTGCAGTTGTCCTCTAGC; F2 GAGCCGGACGTTCTGGTAC; R2 TCCCACACGGAGTTGATGTG; 6xp8: F1 CGTCCAGGTACTTGACCAAC; R1 AGTGTAGCTTTAAACTCCCCGTC; F2 TATCAGGACGTGCTTAAGGAG; R2 GGAGTAGGCACAGGAGTAACATG; This primer set can efficiently amplify the complete genome of the novel HCV subtype 6xp into eight overlapping fragments, thereby obtaining the complete viral genome sequence.

[0007] 2. The primer set of this invention for detecting drug resistance mutation sites of the novel HCV subtype 6xp can specifically amplify the three drug resistance segments NS3, NS5A, and NS5B of the novel HCV subtype 6xp. The nucleotide sequence of the primer set is shown below: NS3-1: F1 CGCAGCATGTGGCGACATC; R1 CCRATSCCMAGCACCGTCGT; F2 TSGGMCCSGCSGATGATGTG; R2 GTCTGTGGAATGGCACTCATC; NS3-2: F1 ATCCATCTGTGGCCGCCAC; R1 GGTCAGCCGACATGCAGGTC; F2 GGAGTTATATGAGCAAAGCCYATGG; R2 ATGTACTTGGTGATGGGAKGTG; NS5A: F1 CTCAGTTCCCTCACCATAACCAG; R1 CTCCAGGCTCACCYTCAAG; F2 CTCCGYAGACTACATGAATGGATC; R2 CATTGAGCTGTAAGAACCGTCG; NS5B-1: F1 TTGARGGTGAGCCTGGAGAC; R1 GTCWCCGCAGACCAACATGTC; F2 GTCATGGTCCACAGTCAGCG; R2 GTCAAAGTCCTTGAGATTGGCTG; NS5B-2: F1 CARGACATGGCGCTCCAAGA; R1 GACAAGTCGAGCTGGGACG; F2 ACCAGATGYTTYGACTCYACTGTCAC; R2 CGCAGTGGAGTGAGTTTGGC.

[0008] The drug resistance mutation sites include V36, T54, R155, A156, D168, Q41, F43, Y56, S122, V158, I170, and M175 in the NS3 region; Q30, L31, Y93, M28, H58, and P58G in the NS5A region; and S282, C316, M414, A421, P495, and S556 in the NS5B region.

[0009] 3. The above full-length amplification primer set or drug resistance mutation detection primer set can be used to prepare HCV new subtype 6xp typing identification reagents or kits or drug resistance mutation site detection reagents or kits.

[0010] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides the first tool for whole-genome amplification of the novel 6xp subtype: The full-length primer set designed in this invention effectively overcomes the problem of low amplification efficiency of existing conventional primer pairs for novel subtypes, and can stably and efficiently obtain the full-length sequence of the novel 6xp subtype, providing key technical support for the phylogenetic analysis and virological research of this novel subtype.

[0011] 2. Comprehensive coverage of drug resistance mutation sites and high detection sensitivity: The drug resistance mutation detection primer set provided by this invention innovatively achieves precise coverage of three dispersed drug resistance regions: NS3, NS5A, and NS5B of the new 6xp subtype. This primer set is highly specific, exhibiting extremely high amplification efficiency even for low-concentration HCV RNA samples, significantly reducing the rate of missed diagnoses and misdiagnoses in clinical testing.

[0012] 3. High clinical translational value: Based on the primer set described in this invention combined with first-generation sequencing technology (Sanger sequencing), it is possible to achieve high-accuracy genotyping and drug resistance mutation analysis of the new HCV 6xp subtype at a low cost, providing direct and reliable molecular diagnostic basis for the clinical development of individualized DAA treatment plans. Attached Figure Description

[0013] Figure 1 The results of agarose gel electrophoresis of the whole genome PCR products in Example 1 are shown. Figure 2 This is a partial phylogenetic diagram of the E1 gene region of the novel HCV subtype 6xp in Example 1; Figure 3 This is a partial phylogenetic diagram of the NS5B gene region of the novel HCV subtype 6xp in Example 1; Figure 4 This is the whole-genome phylogenetic tree of the novel HCV subtype 6xp in Example 1; Figure 5 Example 1: HCV novel subtype 6xp SimPlot recombination analysis graph; Figure 6 Example 1: Genetic distance diagram of the novel HCV subtype 6xp and other genotypes; Figure 7 The results of agarose gel electrophoresis of the PCR products in Example 2 are shown. Detailed Implementation

[0014] To further illustrate the technical means and effects of this invention, the following specific embodiments are provided to further explain the technical solution of this invention. However, this invention is not limited to the scope of these embodiments. The materials used in the following embodiments are not limited to those listed above and may be replaced by other similar materials. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0015] Example 1: Design of full-length primers and acquisition of full-length sequence for the novel HCV subtype 6xp 1. Full-length primer design The E1 and NS5B gene fragments from the three previously obtained novel HCV subtype 6xp samples (sample numbers: YN-HG-1, YN-HG-2, and YN-HG-3) were subjected to BLAST alignment analysis on the NCBI website. Sequences closely related to these gene fragments were downloaded as reference sequences. Subsequently, alignment was performed using Maft software, and primers were designed, evaluated, and optimized using Primer Select software. The final full-length amplification primers meeting the requirements were determined, and their sequences are shown in Table 1. Table 1 Full-length primer sequences for the novel HCV subtype 6xp ; .

[0016] 2. In this embodiment, the commercially available TIANamp Virus RNA Kit (TIANGEN, Beijing) was used to extract viral RNA from three samples of the new subtype 6xp. The extraction method was performed in accordance with the kit's instructions.

[0017] 3. Obtaining the full-length sequence Using the outer primers (F1, R1) in Table 1 and the extracted RNA as a template, the first round of RT-PCR amplification was performed. The reaction system consisted of 1 μL Prime Script 1 Step Enzyme Mix, 12.51 μL Buffer A, 1 μL PrimeF1 (10 μM), 1 μL PrimeR1 (10 μM), 7.5 μL RNase Free H2O, and 2 μL Template (RNA). The reaction conditions were as follows: (a) reverse transcription at 50℃ for 30 min; (b) pre-denaturation at 94℃ for 3 min; (c) denaturation at 94℃ for 30 s, annealing at 50℃ for 30 s, extension at 72℃ for 3 min, for a total of 35 cycles; and (d) extension at 72℃ for 7 min.

[0018] Using the first-round PCR product as a template, the second round of nested PCR amplification was performed using the inner primers (F2, R2) in Table 1.

[0019] The reaction system consisted of 10 μL Green Taq Mix, 6 μL sterile H2O, 1 μL PrimeF2 (10 μM), 1 μL PrimeR2 (10 μM), and 2 μL of the PCR product from the previous step. The reaction conditions were: (a) 94℃ pre-denaturation for 5 min; (b) 94℃ denaturation for 30 s, 50℃ annealing for 30 s, and 72℃ extension for 2 min, for a total of 35 cycles; (c) 72℃ extension for 7 min.

[0020] After the PCR amplification reaction was completed, electrophoresis was performed on a 1% agarose gel. The results showed that the size of each target fragment was consistent with the expected fragment size. Figure 1 As shown. Samples that tested positive for HCV by agarose gel electrophoresis were purified using an OMEGA gel extraction kit. The extracted gel products were stored at -40℃ for later use. The DNA products obtained after gel extraction were sent to Kunming Qingke Biotechnology Co., Ltd. for first-generation sequencing. Because the target fragment was larger than 800 bp, bidirectional sequencing was used to ensure the accuracy of the sequencing results. After obtaining the sequencing results, BLAST alignment was performed on the NCBI website to verify whether the amplified samples were HCV positive. After confirming HCV positivity, the eight fragments obtained from the amplification of each sample were assembled using CExpress software. After assembly, the full-length sequence was corrected using the standard strain H77 to ensure the accuracy and integrity of the sequence, providing a reliable sequence basis for subsequent HCV 6xp subtype typing analysis and drug resistance site detection. The nucleotide sequence of the novel HCV subtype 6xp is shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0021] 4. Validation and Feature Analysis of Full-Length Sequences To verify the accuracy and specificity of the full-length genome sequences amplified by the method described in this invention, phylogenetic analysis, recombination analysis, and genetic distance analysis were performed on the obtained sequences (SEQ ID NO: 1-SEQ ID NO: 3). Specifically, named HCV 6 genotype whole genome sequences were screened and downloaded from the NCBI, VIR, and HCV sequence databases, and unnamed HCV 6 genotype whole genome sequences with sequence integrity ≥95% were also screened and downloaded from the databases. Phylogenetic trees for the E1 gene region, NS5B gene region, and whole genome of HCV 6 genotype were constructed using BioEdit v.7.1.5 software, and the results are as follows: Figure 2 , Figure 3 , Figure 4As shown; Simplot software was used to perform whole-genome Simplot recombination analysis on 6xp and all other HCV type 6 (6a-6xk) sequences to determine whether recombination occurred. The results are as follows. Figure 5 , Figure 6 As shown; using MEGA7, the pairwise genetic distances between the three sequences of 6xp were calculated, and the genetic distances between 6xp and other subtypes 6a-6xk in HCV type 6 were also calculated. The results are as follows. Figure 7 As shown.

[0022] The above analysis results fully confirm that the HCV 6xp subtype full-length amplification primer set and related detection methods described in this invention have good accuracy and reliability, and can stably and accurately obtain the full-length genome sequence of the HCV 6xp subtype, providing reliable technical support and guarantee for the identification of this subtype and subsequent related research.

[0023] Example 2: Detection of the 6xp resistance mutation site of the new HCV subtype 1. Design of primers for detecting drug resistance mutation sites in the novel HCV subtype 6xp The whole genome sequences of HCV type 6 related genes were searched and downloaded from databases such as NCBI, VIR, and the HCV sequence database. These sequences included the whole genome sequences of three novel HCV 6xp subtype strains used in this invention. The sequence selection criteria for primer design were sequence integrity ≥95% and no obvious sequence deletions or mutations. Primers were designed using Primer Select software based on the selected whole genome sequences of HCV type 6 related genes. During the design process, primer design parameters were strictly controlled to avoid the formation of secondary structures within the primers themselves and primer pairing, thereby effectively avoiding non-specific amplification or low amplification efficiency. Simultaneously, primer design strictly adhered to core design principles to ensure that the designed primer set covered all drug-resistant regions of the novel HCV 6xp subtype, specifically including the three functional regions NS3, NS5A, and NS5B. This ensured effective amplification of the target sequences in these three regions and accurate detection of mutations at all drug-resistant target sites within the regions. The nucleotide sequences of the primer set are shown in Table 2. Table 2. Specific primer sequences for drug-resistant regions ; 2. Obtaining the drug resistance segment sequence Using the RNA extracted from samples YN-HG-1, YN-HG-2, and YN-HG-3 in step 2 of Example 1 as templates, the first round of RT-PCR amplification of the drug-resistant region was performed using the outer primers (F1, R1) in Table 2.

[0024] The reaction system consisted of: Prime Script 1 Step Enzyme Mix 1 μL, Buffer A 6 μL, PrimeF1 (10 μM) 1 μL, PrimeR1 (10 μM) 1 μL, and Template (RNA) 3 μL. The reaction conditions were as follows: (a) reverse transcription at 50℃ for 30 min; (b) pre-denaturation at 94℃ for 3 min; (c) denaturation at 94℃ for 30 s, annealing at 50℃ for 30 s, extension at 72℃ for 2 min 30 s, for a total of 35 cycles; and (d) extension at 72℃ for 7 min.

[0025] Using the first-round PCR product of the drug-resistant region as a template, the second round of nested PCR amplification was performed using the inner primers (F2, R2) in Table 4. The reaction system consisted of 10 μL Green Taq Mix, 5 μL sterile H2O, 1 μL PrimeF2 (10 μM), 1 μL PrimeR2 (10 μM), and 3 μL of the PCR product from the previous step. The reaction conditions were as follows: (a) 94℃ pre-denaturation for 5 min; (b) 94℃ denaturation for 30 s, 50℃ annealing for 30 s, and 72℃ extension for 2 min, for a total of 35 cycles; (c) 72℃ extension for 7 min.

[0026] After the PCR reaction, the amplification products were detected by electrophoresis using a 1% agarose gel. The results are as follows: Figure 7 As shown in the figure, the target band can be amplified in all three drug-resistant regions.

[0027] The product was purified using an OMEGA gel extraction kit and then sent to Kunming Qingke Biotechnology Co., Ltd. for first-generation sequencing using an ABI 3730XL automated sequencer. The gene sequences of the three drug-resistant regions of HCV obtained from the sequencing were analyzed for drug resistance sites at http: / / hcv.geno2pheno.org / index.ph. The results are shown in Table 3. Sequencing was successful in 100% (3 / 3) of the samples. Statistically, 33.33% (1 / 3) of the samples showed a mutation at position 155 of the NS3 drug-resistant region; 100% (3 / 3) of the samples showed a mutation at position 122 of the NS3 drug-resistant region; 100% (3 / 3) of the samples showed a mutation at position 58 of the NS5A drug-resistant region; and 100% (3 / 3) of the samples showed a mutation at position 559 of the NS5B drug-resistant region. This experiment shows that the mutation rates at positions 122 of the NS3 resistance region, 58 of the NS5A resistance region, and 559 of the NS5B resistance region are relatively high. Combined with existing research on HCV drug resistance, these three sites are potential key sites affecting the sensitivity of target virus drugs, and their mutations may lead to a decrease in the therapeutic effect of target virus drugs.

[0028] Table 3. Drug resistance mutation sites of the new HCV subtype 6xp .

Claims

1. A set of amplification primers for hepatitis C virus subtypes 6xp, characterized in that, The nucleotide sequences of the primer sets are as follows: 6xp1: F1 GCGACACTCCACCATGATC; R1 TMGCCCARTTAGCCAYCATGC; F2 CCATGATCACTCCCCTGTGAG; R2 ATGCTGTAATATAGCACGCCAAG; 6xp2: F1 TGTGGTGGTGTGTTCCTGGTTG; R1 TTCTCYAGAGCCGCCTCAGC; F2 GACAACTTTTCACCTTCCGG; R2 CCTCAGCCTGAGATATAAGCATCA; 6xp3: F1 TGCTCGTTTACCACTATGCCG; R1 GTGACGATMGTCCCCATMAGG; F2 TTGTCRACCGGYTTAATACATCTAC; R2 TGTTGGTGGTACGCCGTAATG; 6xp4: F1 TGACCTTGCCGTAGCGGTTG; R1 TCAAGGCTRAARTCRACCAYTTG; F2 CGATGGAGAAGAARGTCATCAC; R2 GTCACTGCCACRTTRCARTCAA; 6xp5: F1 GGTAGGCACTTGATCTTCTGTCAC; R1 CTGTTCATCCACTGGTTSGC; F2 AAGAARTGYGACGAGCTGGC; R2 CAGTARGTTGACCATGTCCTCC; 6xp6: F1 ACCCTCCTCCTGAACATCTTAG; R1 CACTTCCTGCCTCCATAGCA; F2 GCWTCTCAGCTTGCWCCAC; R2 TTCGCCTCAAGCAACTCAGC; 6xp7: F1 CTTACCAGTTACGCCATAGGG; R1 TGGTGTGCAGTTGTCCTCTAGC; ​ ​ ​ 2. A primer set for detecting a drug-resistant mutation site of a new subtype 6xp of hepatitis C virus, characterized in that, ​ ​ ​ NS3-2: F1 ATCCATCTGTGGCCGCCAC; R1 GGTCAGCCGACATGCAGGTC; F2 GGAGTTATATGAGCAAAGCCYATGG; R2 ATGTACTTGGTGATGGGAKGTG; NS5A: F1 CTCAGTTCCCTCACCATAACCAG; R1 CTCCAGGCTCACCYTCAAG; F2 CTCCGYAGACTACATGAATGGATC; R2 CATTGAGCTGTAAGAACCGTCG; NS5B-1: F1 TTGARGGTGAGCCTGGAGAC; R1 GTCWCCGCAGACCAACATGTC; F2 GTCATGGTCCACAGTCAGCG; R2 GTCAAAGTCCTTGAGATTGGCTG; NS5B-2: F1 CARGACATGGCGCTCCAAGA; R1 GACAAGTCGAGCTGGGACG; F2 ACCAGATGYTTYGACTCYACTGTCAC; R2 CGCAGTGGAGTGAGTTTGGC.

3. The primer set of claim 2, wherein: The primer set is used to amplify the drug resistance mutation sites in the three drug resistance regions NS3, NS5A and NS5B.

4. The use of the primer set described in claim 1 in the preparation of a reagent or kit for obtaining the whole genome of the novel hepatitis C virus subtype 6xp.

5. The use of the primer set according to claim 2 in the preparation of reagents or kits for detecting the new subtype 6xp drug-resistant mutation of hepatitis C virus.

Images