Establishment of Real Time Allele Specific Locked Nucleic Acid Quantitative PCR for Detection of HBV YIDD (ATT) Mutation and Evaluation of Its Application

<div><p>Background</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>The linear range of the assay was between 1×10⁹ copies/μl and 1×10² copies/μl. The low detection limit was 1×10¹ copies/μl. Sensitivity of the assay were 10⁻⁶, 10⁻⁴ and 10⁻² in the wild-type background of 1×10⁹ copies/μl, 1×10⁷ copies/μl and 1×10⁵ 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 (<i>Kappa</i> = 0.676, <i>P</i> = 0.000). Minor variants can be detected 18 weeks earlier than the rebound of HBV DNA load and alanine aminotransferase level.</p><p>Conclusions</p><p>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.</p></div

The correlation between actual and calculated proportion of mutant plasmid DNA at the concentration of 1×10⁷ copies/μl.

<p><i>R²</i> = 0.9907, <i>P</i><0.0001.</p

Amplification plot and standard curve of RT-AS-LNA-qPCR for pMD-18-YMDD.

<p>(A) Amplification plot with different colors represented different concentrations of pMD-18-YMDD plasmids which were illustrated in the figure. (B) Standard curve of pMD-18-YMDD: Y = -3.478X+41.654 (<i>R²</i> = 0.999).</p

Dynamics of HBV YMDD, YIDD DNA levels and serum ALT level during LMV and LMV + ADV therapy in a CHB patient with breakthrough hepatitis.

<p>Dynamics of HBV YMDD, YIDD DNA levels and serum ALT level during LMV and LMV + ADV therapy in a CHB patient with breakthrough hepatitis.</p

Detection sensitivity comparison of RT-AS-LNA-qPCR and sequencing on clinical samples containing different proportions of rtM204I variant.

<p>M: Methionine; I: isoleucine; -: not performed; NTC: no-template control; N: undetectable.</p

Clinical templates with different proportions of mutant DNA were tested by mutant specific primer set.

<p>Amplification plot with different colors represented different proportions of mutant DNA which were illustrated in the figure. The Ct of no-template control was undetectable.</p

Concordance between RT-AS-LNA-qPCR and direct sequencing.

<p>Complete concordant results are shown in bold. Partial concordant results are shown in italics.</p

Partial concordant results between RT-AS-LNA-qPCR and direct sequencing.

<p>Partial concordant results between RT-AS-LNA-qPCR and direct sequencing.</p

Primers for RT-AS-LNA-qPCR.

<p>KF/KF_n indicate the common forward primers to all reactions. L1/L2/L2_l/L1_n/L2_n indicate specific RT-AS-LNA-qPCR reverse primers. M indicates A/C; K indicates G/T; W indicates A/T; +A/+C indicate LNA nucleosides.</p

Typical chromatograms of the cloning sequencing of HBV rtM204 and rtM204I.

<p>(A) Chromatograms of rtM204 (wild-type). The bases in 204 site were ATG, indicated by the arrow. (B) Chromatograms of rtM204I (mutant). The bases in 204 site were ATT, indicated by the arrow.</p