Sensitiviy of pyrosequencing and PCR product direct sequencing in detection of drug resistance genes in hepatitis B virus:a comparative study
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OBJECTIVE To evaluate the potential value of pyrosequencing method in the detection of HBV resistance genes through the comparison with PCR product direct sequencing method.METHODS The patients with chronic hepatitis B treated with lamivudine were selected,the sensitivity and specificity of two methods mentioned above were compared by detecting the variation of lamivudine resistance mutation site rt180aa.RESULTS Both pyrosequencing and Sanger sequencing could detect the dominant virus(90%) exactly.When the proportion of rt180 mutation ranged from 90% to 50%,which was detected by pyrosequencing,the detection value by Sanger sequencing were reduced to 76.92% and 50.00%;when the proportion value of rt180M was within 50%-10% determined by pyrosequencing,it would be reduced to 10.00%-15.38% by Sanger sequencing method;that means quasispecies less than 10% could be only detected by pyrosequencing rather than by Sanger sequencing.CONCLUSION As compared with the Sanger sequencing,pyrosequencing method is with better sensitivity in the detection of variation of hepatitis B virus,which can detect the quasispecies and its population drift during the antiviral therapy process,with great significance in research on the antiviral effect and the forecast of drug resistance.Keywords:
Pyrosequencing
Sanger sequencing
Viral quasispecies
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Detection of mutations associated to nucleos(t)ide analogs and hepatitis B virus (HBV) genotyping are essential for monitoring treatment of HBV infection. We developed a multiplex polymerase chain reaction-ligase detection reaction (PCR-LDR) assay for the rapid detection of HBV genotypes and mutations associated with lamivudine, adefovir, and telbivudine resistance in HBV-infected patients. HBV templates were amplified by PCR, followed by LDR and electrophoresis on a sequencer. The assay was evaluated using plasmids that contained wild-type or mutant HBV sequences and 216 clinical samples. The PCR-LDR assay and sequencing gave comparable results for 158 of the 216 samples (73.1%) with respect to mutation detection and genotyping. Complete agreement between the two methods was observed for all the samples (100%) at codon 180 and codon 204. Concordant results were observed for 99.4% of the 158 samples at codon 181 and 98.7% at codon 236. The genotyping results were completely concordant between the PCR-LDR assay and sequencing. The PCR-LDR assay could detect a proportion of 1% mutant plasmid in a background of wild-type plasmid. The PCR-LDR assay is sensitive and specific for detection of HBV genotypes and drug resistance mutations, and could be helpful for decision making in the treatment of HBV infection.
Telbivudine
Adefovir
Ligase chain reaction
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Objective To investigate the feasibility of dual-probe real-time PCR assay for detection of lamivudine-resistance hepatitis B virus.Methods MGB probes with different fluorescences were designed and synthesized.The serum samples of hepatitis B patients treated with lamivudine were detected by real-time PCR and sequencing,and the results were compared.Results In total 68 serum samples,27 samples are YMDD,20 YIDD and 11 YVDD,which was consistent with that of sequencing(85.3%).10 samples were detected to be mixed population.Further sequencing assay after cloning demonstrated that the direct sequencing method could only give sequence of major virus in mixed population.Conclusions MGB dual-probe real-time PCR assay can quickly and accurately detect HBV YMDD mutations especially in mixed population.
Cloning (programming)
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ABSTRACT Mutations in the highly conserved tyrosine-methionine-aspartate-aspartate (YMDD) motif are frequently associated with resistance to antivirals and represent a major concern in the treatment of hepatitis B virus (HBV) infection. Conventional methods fail to detect minority populations of drug-resistant viral quasispecies if they represent less than 25% of the total sample virus population. The amplification refractory mutation system real-time PCR (ARMS RT-PCR) was combined with molecular beacon technology using the LightCycler system. The samples from HBV patients selected for assay evaluation included (i) 57 samples from treatment-naïve patients for biological discriminatory ability (cutoff) estimation, (ii) 12 samples from patients with treatment failure that were M204V positive by sequencing, and (iii) 13 samples from patients with treatment failure that were negative for mutation at codon 204 by sequencing. The discriminatory ability of the assay was 0.25% when tested with laboratory-synthesized DNA target sequences. The median mutant-to-wild-type ratio for samples from naive patients tested positive for the wild type and for mutant variants was 0.01% (5th and 95th percentiles = 0.0001 and 0.04%, respectively). A value of 0.04% was selected as the biological cutoff of the assay of clinical samples. In all samples M204V positive by sequencing (12/12), the mutant variant was detected as the predominant population (range, 82.76 to 99.43%). Interestingly, in 5 (38%) of 13 samples negative by sequencing, the M204V variant was detected at a ratio above the biological cutoff (0.05 to 28%). The assay represents an efficient technique for the early detection and quantification of M204V variants before mutant strains emerge to dominate the population.
Viral quasispecies
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Background Ultra deep sequencing is of increasing use not only in research but also in diagnostics. For implementation of ultra deep sequencing assays in clinical laboratories for routine diagnostics, intra- and inter-laboratory testing are of the utmost importance. Methods A multicenter study was conducted to validate an updated assay design for 454 Life Sciences’ GS FLX Titanium system targeting protease/reverse transcriptase (RTP) and env (V3) regions to identify HIV-1 drug-resistance mutations and determine co-receptor use with high sensitivity. The study included 30 HIV-1 subtype B and 6 subtype non-B samples with viral titers (VT) of 3,940–447,400 copies/mL, two dilution series (52,129–1,340 and 25,130–734 copies/mL), and triplicate samples. Amplicons spanning PR codons 10–99, RT codons 1–251 and the entire V3 region were generated using barcoded primers. Analysis was performed using the GS Amplicon Variant Analyzer and geno2pheno for tropism. For comparison, population sequencing was performed using the ViroSeq HIV-1 genotyping system. Results The median sequencing depth across the 11 sites was 1,829 reads per position for RTP (IQR 592–3,488) and 2,410 for V3 (IQR 786–3,695). 10 preselected drug resistant variants were measured across sites and showed high inter-laboratory correlation across all sites with data (P<0.001). The triplicate samples of a plasmid mixture confirmed the high inter-laboratory consistency (mean% ± stdev: 4.6 ±0.5, 4.8 ±0.4, 4.9 ±0.3) and revealed good intra-laboratory consistency (mean% range ± stdev range: 4.2–5.2 ± 0.04–0.65). In the two dilutions series, no variants >20% were missed, variants 2–10% were detected at most sites (even at low VT), and variants 1–2% were detected by some sites. All mutations detected by population sequencing were also detected by UDS. Conclusions This assay design results in an accurate and reproducible approach to analyze HIV-1 mutant spectra, even at variant frequencies well below those routinely detectable by population sequencing.
Amplicon
Pyrosequencing
Amplicon sequencing
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Objective: To establish the method of detection hepetitis B virus drug-resistance gene by nested PCR combined with pyrosequencing.Methods: For the upstream and downstream of 8 drug-resistance site of HBV P gene RT region,designing two specific primer pairs,building the nested PCR amplification system.And using this method combining with pyrosequencing to test the 8 drug-resistance site and the HBV genotype of 223 samples from chronic hepatitis B patients.Results: The detection limit of this nested PCR combining pyrosequencing study was 2 × 103copies / ml,and the positive bands can be amplified from each clinical sample.In 192 cases(treatment group) using nucleoside analogues,153(79.7%) with genotype B,39(20.3%) with genotype C,53(27.6%) had drug-resistance mutation,with 30.1%(16 cases) 204-site mutation,and 5.6%(3 /53) both in 180-site and 181-site,3.8%(2 /53) with 236-site mutation,1.9%(1 /53) with 202-site mutation;37.7%(20 /53) had both 180204 site mutation,and 3.8%(2 /53) with 181 /236 site mutation and 204 250 site mutation;3 sites mutation in the same time with the percentage 7.5%(173 180 240、180 181 204、180 184 204、 180 202 204 in one case) 。In the 31 cases of patients from untreated group,25(80.6%) with genotype B,6(19.4%) with genotype C,none drug resistance was founded.Conclusion: This method can be used for both hepatitis B virus genotyping and drug-resistance site mutation detection,also can guide drug selection and therapeutic evaluation,is very meaningful for clinical use.
Pyrosequencing
Primer (cosmetics)
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ObjectivesHIV drug resistance is a major concern as the emergence of resistant strains of virus results in failure of first-line therapies with an associated increase in the cost of subsequent regimens. Genotypic resistance is currently assessed by direct sequencing and cannot detect resistant species below 20%. Real-time PCR amplification was assessed for its ability to detect the signature mutation for nelfinavir, D30N.
Nelfinavir
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Pyrosequencing
Nevirapine
Variants of PCR
Concordance
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ABSTRACT Global HIV treatment programs need sensitive and affordable tests to monitor HIV drug resistance. We compared mutant detection by the oligonucleotide ligation assay (OLA), an economical and simple test, to massively parallel sequencing. Nonnucleoside reverse transcriptase inhibitor (K103N, V106M, Y181C, and G190A) and lamivudine (M184V) resistance mutations were quantified in blood-derived plasma RNA and cell DNA specimens by OLA and 454 pyrosequencing. A median of 1,000 HIV DNA or RNA templates (range, 163 to 1,874 templates) from blood specimens collected in Mozambique ( n = 60) and Kenya ( n = 51) were analyzed at 4 codons in each sample ( n = 441 codons assessed). Mutations were detected at 75 (17%) codons by OLA sensitive to 2.0%, at 71 codons (16%; P = 0.78) by pyrosequencing using a cutoff value of ≥2.0%, and at 125 codons (28%; P < 0.0001) by pyrosequencing sensitive to 0.1%. Discrepancies between the assays included 15 codons with mutant concentrations of ∼2%, one at 8.8% by pyrosequencing and not detected by OLA, and one at 69% by OLA and not detected by pyrosequencing. The latter two cases were associated with genetic polymorphisms in the regions critical for ligation of the OLA probes and pyrosequencing primers, respectively. Overall, mutant concentrations quantified by the two methods correlated well across the codons tested ( R 2 > 0.8). Repeat pyrosequencing of 13 specimens showed reproducible detection of 5/24 mutations at <2% and 6/6 at ≥2%. In conclusion, the OLA and pyrosequencing performed similarly in the quantification of nonnucleoside reverse transcriptase inhibitor and lamivudine mutations present at >2% of the viral population in clinical specimens. While pyrosequencing was more sensitive, detection of mutants below 2% was not reproducible.
Pyrosequencing
Massive parallel sequencing
Stop codon
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Sanger sequencing
Foscarnet
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