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Basic Characteristics of Mutations
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Mutation Site
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M204I |
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Mutation Site Sentence
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The rtM204I (ATT coding for isoleucine) is one of the most important resistance mutation sites. |
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Mutation Level
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Amino acid level |
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Mutation Type
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Nonsynonymous substitution |
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Gene/Protein/Region
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RT |
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Standardized Encoding Gene
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P
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Genotype/Subtype
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- |
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Viral Reference
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-
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Functional Impact and Mechanisms
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Disease
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Hepatitis B Virus Infection
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Immune
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- |
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Target Gene
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-
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Clinical and Epidemiological Correlations
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Clinical Information
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- |
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Treatment
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- |
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Location
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China |
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Literature Information
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PMID
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24587198
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Title
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Establishment of real time allele specific locked nucleic acid quantitative PCR for detection of HBV YIDD (ATT) mutation and evaluation of its application
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Author
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Zeng Y,Li D,Wang W,Su M,Lin J,Chen H,Jiang L,Chen J,Yang B,Ou Q
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Journal
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PloS one
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Journal Info
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2014 Feb 28;9(2):e90029
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Abstract
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BACKGROUND: Long-term use of nucleos(t)ide analogues can increase risk of HBV drug-resistance mutations. The rtM204I (ATT coding for isoleucine) is one of the most important resistance mutation sites. Establishing a simple, rapid, reliable and highly sensitive assay to detect the resistant mutants as early as possible is of great clinical significance. METHODS: Recombinant plasmids for HBV YMDD (tyrosine-methionine-aspartate-aspartate) and YIDD (tyrosine-isoleucine-aspartate-aspartate) were constructed by TA cloning. Real time allele specific locked nucleic acid quantitative PCR (RT-AS-LNA-qPCR) with SYBR Green I was established by LNA-modified primers and evaluated with standard recombinant plasmids, clinical templates (the clinical wild type and mutant HBV DNA mixture) and 102 serum samples from nucleos(t)ide analogues-experienced patients. The serum samples from a chronic hepatitis B (CHB) patient firstly received LMV mono therapy and then switched to LMV + ADV combined therapy were also dynamically analyzed for 10 times. RESULTS: The linear range of the assay was between 1x10(9) copies/mul and 1 x 10(2) copies/mul. The low detection limit was 1 x 10(1) copies/mul. Sensitivity of the assay were 10(-6), 10(-4) and 10(-2) in the wild-type background of 1 x 10(9) copies/mul, 1 x 10(7) copies/mul and 1 x 10(5) copies/mul, respectively. The sensitivity of the assay in detection of clinical samples was 0.03%. The complete coincidence rate between RT-AS-LNA-qPCR and direct sequencing was 91.2% (93/102), partial coincidence rate was 8.8% (9/102), and no complete discordance was observed. The two assays showed a high concordance (Kappa = 0.676, P = 0.000). Minor variants can be detected 18 weeks earlier than the rebound of HBV DNA load and alanine aminotransferase level. CONCLUSIONS: A rapid, cost-effective, high sensitive, specific and reliable method of RT-AS-LNA-qPCR with SYBR Green I for early and absolute quantification of HBV YIDD (ATT coding for isoleucine) variants was established, which can provide valuable information for clinical antiretroviral regimens.
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Sequence Data
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-
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