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Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing
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| Zeitschriftentitel: | Journal of Clinical Microbiology |
|---|---|
| Personen und Körperschaften: | , , , , , , , , |
| In: | Journal of Clinical Microbiology, 52, 2014, 8, S. 2933-2939 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Society for Microbiology
|
| Schlagwörter: |
| author_facet |
Liu, Can Lin, Jinpiao Chen, Huijuan Shang, Hongyan Jiang, Ling Chen, Jing Ye, Yang Yang, Bin Ou, Qishui Liu, Can Lin, Jinpiao Chen, Huijuan Shang, Hongyan Jiang, Ling Chen, Jing Ye, Yang Yang, Bin Ou, Qishui |
|---|---|
| author |
Liu, Can Lin, Jinpiao Chen, Huijuan Shang, Hongyan Jiang, Ling Chen, Jing Ye, Yang Yang, Bin Ou, Qishui |
| spellingShingle |
Liu, Can Lin, Jinpiao Chen, Huijuan Shang, Hongyan Jiang, Ling Chen, Jing Ye, Yang Yang, Bin Ou, Qishui Journal of Clinical Microbiology Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing Microbiology (medical) |
| author_sort |
liu, can |
| spelling |
Liu, Can Lin, Jinpiao Chen, Huijuan Shang, Hongyan Jiang, Ling Chen, Jing Ye, Yang Yang, Bin Ou, Qishui 0095-1137 1098-660X American Society for Microbiology Microbiology (medical) http://dx.doi.org/10.1128/jcm.01127-14 <jats:title>ABSTRACT</jats:title> <jats:p> 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 ( <jats:italic> T <jats:sub>c</jats:sub> </jats:italic> ) was established as a denaturing temperature for both fast and full COLD-PCR procedures. For single mutations, when a melting temperature ( <jats:italic> T <jats:sub>m</jats:sub> </jats:italic> )-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 <jats:italic> T <jats:sub>m</jats:sub> </jats:italic> (e.g., C-G→G-C) or raised <jats:italic> T <jats:sub>m</jats:sub> </jats:italic> (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. </jats:p> Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing Journal of Clinical Microbiology |
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| title |
Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing |
| title_unstemmed |
Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing |
| title_full |
Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing |
| title_fullStr |
Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing |
| title_full_unstemmed |
Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing |
| title_short |
Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing |
| title_sort |
detection of hepatitis b virus genotypic resistance mutations by coamplification at lower denaturation temperature-pcr coupled with sanger sequencing |
| topic |
Microbiology (medical) |
| url |
http://dx.doi.org/10.1128/jcm.01127-14 |
| publishDate |
2014 |
| physical |
2933-2939 |
| description |
<jats:title>ABSTRACT</jats:title>
<jats:p>
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 (
<jats:italic>
T
<jats:sub>c</jats:sub>
</jats:italic>
) was established as a denaturing temperature for both fast and full COLD-PCR procedures. For single mutations, when a melting temperature (
<jats:italic>
T
<jats:sub>m</jats:sub>
</jats:italic>
)-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
<jats:italic>
T
<jats:sub>m</jats:sub>
</jats:italic>
(e.g., C-G→G-C) or raised
<jats:italic>
T
<jats:sub>m</jats:sub>
</jats:italic>
(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.
</jats:p> |
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| author | Liu, Can, Lin, Jinpiao, Chen, Huijuan, Shang, Hongyan, Jiang, Ling, Chen, Jing, Ye, Yang, Yang, Bin, Ou, Qishui |
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| description | <jats:title>ABSTRACT</jats:title> <jats:p> 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 ( <jats:italic> T <jats:sub>c</jats:sub> </jats:italic> ) was established as a denaturing temperature for both fast and full COLD-PCR procedures. For single mutations, when a melting temperature ( <jats:italic> T <jats:sub>m</jats:sub> </jats:italic> )-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 <jats:italic> T <jats:sub>m</jats:sub> </jats:italic> (e.g., C-G→G-C) or raised <jats:italic> T <jats:sub>m</jats:sub> </jats:italic> (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. </jats:p> |
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| spelling | Liu, Can Lin, Jinpiao Chen, Huijuan Shang, Hongyan Jiang, Ling Chen, Jing Ye, Yang Yang, Bin Ou, Qishui 0095-1137 1098-660X American Society for Microbiology Microbiology (medical) http://dx.doi.org/10.1128/jcm.01127-14 <jats:title>ABSTRACT</jats:title> <jats:p> 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 ( <jats:italic> T <jats:sub>c</jats:sub> </jats:italic> ) was established as a denaturing temperature for both fast and full COLD-PCR procedures. For single mutations, when a melting temperature ( <jats:italic> T <jats:sub>m</jats:sub> </jats:italic> )-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 <jats:italic> T <jats:sub>m</jats:sub> </jats:italic> (e.g., C-G→G-C) or raised <jats:italic> T <jats:sub>m</jats:sub> </jats:italic> (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. </jats:p> Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing Journal of Clinical Microbiology |
| spellingShingle | Liu, Can, Lin, Jinpiao, Chen, Huijuan, Shang, Hongyan, Jiang, Ling, Chen, Jing, Ye, Yang, Yang, Bin, Ou, Qishui, Journal of Clinical Microbiology, Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing, Microbiology (medical) |
| title | Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing |
| title_full | Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing |
| title_fullStr | Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing |
| title_full_unstemmed | Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing |
| title_short | Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing |
| title_sort | detection of hepatitis b virus genotypic resistance mutations by coamplification at lower denaturation temperature-pcr coupled with sanger sequencing |
| title_unstemmed | Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing |
| topic | Microbiology (medical) |
| url | http://dx.doi.org/10.1128/jcm.01127-14 |