NDLI: Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing

Content Provider	PubMed Central
Author	Liu, Can Lin, Jinpiao Chen, Huijuan Shang, Hongyan Jiang, Ling Chen, Jing Yang, Ye Yang, Bin Ou, Qishui
Editor	Tang, Y. W.
Copyright Year	2014
Abstract	Mutations in the reverse transcriptase (rt) region of the DNA polymerase gene are the primary cause of hepatitis B virus (HBV) drug resistance. In this study, we established a novel method that couples coamplification at lower denaturation temperature (COLD)-PCR and Sanger sequencing, and we applied it to the detection of known and unknown HBV mutations. Primers were designed based on the common mutations in the HBV rt sequence at positions 180 to 215. The critical denaturation temperature (Tc ) was established as a denaturing temperature for both fast and full COLD-PCR procedures. For single mutations, when a melting temperature (Tm )-reducing mutation occurred (e.g., C-G→T-A), the sensitivities of fast and full COLD-PCR for mutant detection were 1% and 2%, respectively; when the mutation caused no change in Tm (e.g., C-G→G-C) or raised Tm (e.g., T-A→C-G), only full COLD-PCR improved the sensitivity for mutant detection (2%). For combination mutations, the sensitivities of both full and fast COLD-PCR were increased to 0.5%. The limits of detection for fast and full COLD-PCR were 50 IU/ml and 100 IU/ml, respectively. In 30 chronic hepatitis B (CHB) cases, no mutations were detected by conventional PCR, whereas 18 mutations were successfully detected by COLD-PCR, including low-prevalence mutations (<10%), as confirmed by ultradeep pyrosequencing. In conclusion, COLD-PCR provides a highly sensitive, simple, inexpensive, and practical tool for significantly improving amplification efficacy and detecting low-level mutations in clinical CHB cases.
Related Links	http://dx.doi.org/10.1128/jcm.01127-14
Ending Page	2939
Page Count	7
Starting Page	2933
File Format	PDF
ISSN	00951137
e-ISSN	1098660X
Journal	Journal of Clinical Microbiology
Issue Number	8
Volume Number	52
Language	English
Publisher	American Society for Microbiology
Publisher Date	2014-08-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Microbiology (medical) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Microbiology (medical)

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3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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