title: Establishment of real time allele specific locked nucleic acid quantitative PCR for detection of HBV YIDD (ATT) mutation and evaluation of its application.
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.
authors.affiliation: First Clinical College, Fujian Medical University, Fuzhou, China ; Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors.forename: Yongbin
authors.initials: Y
authors.lastname: Zeng
authors.affiliation: Department of Laboratory Medicine, The Armed Police Hospital of Fujian, Fuzhou, China.
authors.forename: Dezhong
authors.initials: D
authors.lastname: Li
authors.affiliation: First Clinical College, Fujian Medical University, Fuzhou, China ; Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: First Clinical College, Fujian Medical University, Fuzhou, China ; Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors.forename: Mingkuan
authors.initials: M
authors.lastname: Su
authors.affiliation: First Clinical College, Fujian Medical University, Fuzhou, China ; Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors.forename: Jinpiao
authors.initials: J
authors.lastname: Lin
authors.affiliation: First Clinical College, Fujian Medical University, Fuzhou, China ; Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Chen
authors.affiliation: First Clinical College, Fujian Medical University, Fuzhou, China ; Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Jiang
authors.affiliation: First Clinical College, Fujian Medical University, Fuzhou, China ; Center of Liver Diseases, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.affiliation: First Clinical College, Fujian Medical University, Fuzhou, China ; Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
authors.affiliation: First Clinical College, Fujian Medical University, Fuzhou, China ; Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
authors.forename: Qishui
authors.initials: Q
authors.lastname: Ou
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000627:therapeutic use; D000328:Adult; D000483:Alleles; D000998:Antiviral Agents / Q000627:therapeutic use; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D024882:Drug Resistance, Viral / Q000187:drug effects / Q000235:genetics*; D042241:Early Diagnosis; D005260:Female; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D019694:Hepatitis B, Chronic / Q000175:diagnosis* / Q000188:drug therapy / Q000821:virology; D006801:Humans; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D019259:Lamivudine / Q000627:therapeutic use; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D059372:Nucleotide Motifs; D009841:Oligonucleotides / Q000737:chemistry*; D063065:Organophosphonates / Q000627:therapeutic use; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D000998:Antiviral Agents; D017931:DNA Primers; D004279:DNA, Viral; D009841:Oligonucleotides; D063065:Organophosphonates; C477371:locked nucleic acid; D007532:Isoleucine; D019259:Lamivudine; D000225:Adenine; C106812:adefovir dipivoxil