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PLoS One. 2014 Feb 28;9(2):e90029. doi: 10.1371/journal.pone.0090029. eCollection 2014.

Establishment of real time allele specific locked nucleic acid quantitative PCR for detection of HBV YIDD (ATT) mutation and evaluation of its application.

Author information

1
First Clinical College, Fujian Medical University, Fuzhou, China ; Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
2
Department of Laboratory Medicine, The Armed Police Hospital of Fujian, Fuzhou, China.
3
First Clinical College, Fujian Medical University, Fuzhou, China ; Center of Liver Diseases, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.

Abstract

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 1×10(9) copies/μl and 1 × 10(2) copies/μl. The low detection limit was 1 × 10(1) copies/μl. Sensitivity of the assay were 10(-6), 10(-4) and 10(-2) in the wild-type background of 1 × 10(9) copies/μl, 1 × 10(7) copies/μl and 1 × 10(5) copies/μl, 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.

PMID:
24587198
PMCID:
PMC3938556
DOI:
10.1371/journal.pone.0090029
[Indexed for MEDLINE]
Free PMC Article
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