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

Specific locations at which specimens were collected in this study.

<p>★locations of zoos.</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

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

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

Neighbor-joining tree of <i>E</i>. <i>bieneusi</i> ITS genotypes.

<p>Phylogenetic relationship of <i>E</i>. <i>bieneusi</i> ITS nucleotide sequences of this study and other genotypes previously deposited in GenBank, as inferred by a neighbor-joining analysis (software Mega 5, <a href="http://www.megasoftware.net/" target="_blank">http://www.megasoftware.net/</a>) based on genetic distances calculated using the Kimura two-parameter model. The ITS tree was rooted with GenBank sequence DQ885585. Bootstrap values greater than 50% from 1,000 are shown on nodes. Each sequence from GenBank is identified by the accession number, host origin, and the genotype designation. The group terminology for the clusters is based on the works of Thellier and Breton [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117991#pone.0117991.ref021" target="_blank">21</a>], Li et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117991#pone.0117991.ref009" target="_blank">9</a>], and Karim et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117991#pone.0117991.ref003" target="_blank">3</a>]. Two unique sequences of new genotype CM18 and known genotype CM4 in this study are designated as group 9 sequences. Known and new genotypes identified in this study are indicated by open and filled triangles, respectively.</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

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

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

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