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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenine / analogs & derivatives
  • Adenine / therapeutic use
  • Adult
  • Alleles
  • Antiviral Agents / therapeutic use
  • DNA Primers / chemistry
  • DNA, Viral / genetics*
  • DNA, Viral / isolation & purification
  • Drug Resistance, Viral / drug effects
  • Drug Resistance, Viral / genetics*
  • Early Diagnosis
  • Female
  • Hepatitis B virus / drug effects
  • Hepatitis B virus / genetics*
  • Hepatitis B virus / isolation & purification
  • Hepatitis B, Chronic / diagnosis*
  • Hepatitis B, Chronic / drug therapy
  • Hepatitis B, Chronic / virology
  • Humans
  • Isoleucine / genetics
  • Isoleucine / metabolism
  • Lamivudine / therapeutic use
  • Limit of Detection
  • Male
  • Middle Aged
  • Molecular Sequence Data
  • Mutation
  • Nucleotide Motifs
  • Oligonucleotides / chemistry*
  • Organophosphonates / therapeutic use
  • Real-Time Polymerase Chain Reaction / methods*

Substances

  • Antiviral Agents
  • DNA Primers
  • DNA, Viral
  • Oligonucleotides
  • Organophosphonates
  • locked nucleic acid
  • Isoleucine
  • Lamivudine
  • Adenine
  • adefovir dipivoxil

Grants and funding

The study was supported by the grants from Key Project of Industry-university Cooperation of Fujian province (2013Y4002), Social Key Project of Fujian Technology Department (2011Y0023; http://www.fjkjt.gov.cn/) and National Natural Science Foundation of China (81371888). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.