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Article: Application of coamplification at lower denaturation temperature-PCR sequencing for early detection of antiviral drug resistance mutations of hepatitis B virus

TitleApplication of coamplification at lower denaturation temperature-PCR sequencing for early detection of antiviral drug resistance mutations of hepatitis B virus
Authors
Issue Date2014
Citation
Journal of Clinical Microbiology, 2014, v. 52 n. 9, p. 3209-3215 How to Cite?
AbstractNucleoside/nucleotide analogue for the treatment of chronic hepatitis B virus (HBV) infection is hampered by the emergence of drug resistance mutations. Conventional PCR-sequencing cannot detect minor variants of <20%. We developed a modified CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) method for the detection of HBV minority drug resistance mutations. The critical denaturation temperature for COLD-PCR was determined to be 78°C. Sensitivity of COLD-PCR sequencing was determined using serially-diluted plasmids containing mixed proportions of HBV reverse transcriptase (rt) wild-type and mutant sequences. Conventional PCR-sequencing detected mutations only if they existed in ≥25%, whereas COLD-PCR sequencing detected mutations when they existed in 5-10% of the viral population. The performance of COLD-PCR was compared to conventional PCR-sequencing and a line probe (LiPA) assay, using 215 samples obtained from 136 lamivudine- or telbivudine-treated patients with virological breakthrough. Among these 215 samples, drug resistance mutations were detected in 155 (72 %), 148 (69 %) and 113 samples (53 %) by LiPA, COLD-PCR, and conventional PCR-sequencing, respectively. Nineteen (9 %) samples had mutations detectable by COLD-PCR but not LiPA, while 26 (12 %) samples had mutations detectable by LiPA but not COLD-PCR, indicating both methods were comparable (P = 0.371). COLD-PCR was more sensitive than conventional PCR-sequencing: 35 (16 %) samples had mutations detectable by COLD-PCR but not conventional PCR-sequencing, while none had mutations detected by conventional PCR-sequencing but not COLD-PCR (P < 0.0001). COLD-PCR sequencing is a simple method which is comparable to LiPA and superior to conventional PCR-sequencing in detecting minor lamivudine/telbivudine resistance mutations.
Persistent Identifierhttp://hdl.handle.net/10722/198546
PubMed Central ID

 

DC FieldValueLanguage
dc.contributor.authorWong, DKHen_US
dc.contributor.authorTsoi, YYOen_US
dc.contributor.authorHuang, FYen_US
dc.contributor.authorSeto, WKWen_US
dc.contributor.authorFung, JYYen_US
dc.contributor.authorLai, CLen_US
dc.contributor.authorYuen, RMFen_US
dc.date.accessioned2014-07-07T07:17:11Z-
dc.date.available2014-07-07T07:17:11Z-
dc.date.issued2014-
dc.identifier.citationJournal of Clinical Microbiology, 2014, v. 52 n. 9, p. 3209-3215en_US
dc.identifier.urihttp://hdl.handle.net/10722/198546-
dc.description.abstractNucleoside/nucleotide analogue for the treatment of chronic hepatitis B virus (HBV) infection is hampered by the emergence of drug resistance mutations. Conventional PCR-sequencing cannot detect minor variants of <20%. We developed a modified CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) method for the detection of HBV minority drug resistance mutations. The critical denaturation temperature for COLD-PCR was determined to be 78°C. Sensitivity of COLD-PCR sequencing was determined using serially-diluted plasmids containing mixed proportions of HBV reverse transcriptase (rt) wild-type and mutant sequences. Conventional PCR-sequencing detected mutations only if they existed in ≥25%, whereas COLD-PCR sequencing detected mutations when they existed in 5-10% of the viral population. The performance of COLD-PCR was compared to conventional PCR-sequencing and a line probe (LiPA) assay, using 215 samples obtained from 136 lamivudine- or telbivudine-treated patients with virological breakthrough. Among these 215 samples, drug resistance mutations were detected in 155 (72 %), 148 (69 %) and 113 samples (53 %) by LiPA, COLD-PCR, and conventional PCR-sequencing, respectively. Nineteen (9 %) samples had mutations detectable by COLD-PCR but not LiPA, while 26 (12 %) samples had mutations detectable by LiPA but not COLD-PCR, indicating both methods were comparable (P = 0.371). COLD-PCR was more sensitive than conventional PCR-sequencing: 35 (16 %) samples had mutations detectable by COLD-PCR but not conventional PCR-sequencing, while none had mutations detected by conventional PCR-sequencing but not COLD-PCR (P < 0.0001). COLD-PCR sequencing is a simple method which is comparable to LiPA and superior to conventional PCR-sequencing in detecting minor lamivudine/telbivudine resistance mutations.en_US
dc.languageengen_US
dc.relation.ispartofJournal of Clinical Microbiologyen_US
dc.titleApplication of coamplification at lower denaturation temperature-PCR sequencing for early detection of antiviral drug resistance mutations of hepatitis B virusen_US
dc.typeArticleen_US
dc.identifier.emailWong, DKH: danywong@hku.hken_US
dc.identifier.emailTsoi, YYO: otsoi@hku.hken_US
dc.identifier.emailHuang, FY: camy@graduate.hku.hken_US
dc.identifier.emailSeto, WKW: wkseto2@hku.hken_US
dc.identifier.emailFung, JYY: jfung@hkucc.hku.hken_US
dc.identifier.emailLai, CL: hrmelcl@hku.hken_US
dc.identifier.emailYuen, RMF: mfyuen@hku.hken_US
dc.identifier.authorityWong, DKH=rp00492en_US
dc.identifier.authoritySeto, WKW=rp01659en_US
dc.identifier.authorityFung, JYY=rp00518en_US
dc.identifier.authorityYuen, RMF=rp00479en_US
dc.identifier.doi10.1128/JCM.00343-14-
dc.identifier.pmcidPMC4313139-
dc.identifier.hkuros230126en_US

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