Use of Real-Time PCR and Fluorimetry To Detect Lamivudine Resistance-Associated Mutations in Hepatitis B Virus

Abstract

ABSTRACT Very rapid amplification of DNA by PCR in small volumes can be continuously monitored by the detection of the binding of probes with a rapid cycler with built-in fluorometric detection. Primers were designed to amplify approximately 100 bp of the polymerase gene of hepatitis B virus (HBV) spanning codon 550, where mutations associated with resistance to lamivudine invariably occur. Four hybridization probes were synthesized: one was 3′ labelled with fluorescein and hybridized upstream of codon 550. The others were 5′ labelled with Cy5 and 3′ labelled with biotin and spanned codon 550. The Cy5-labelled oligonucleotides contained either wild-type (ATG) or mutant (GTG or ATT) sequences. A Cy5-labelled probe and either the fluorescein-labelled probe or Sybr Green 1 (a compound that fluoresces when bound to double-stranded DNA) were included in each PCR. After completion of the amplification by using a LightCycler (Idaho Technology), the temperature at which the Cy5 probe melted from the product was determined in a melt program that took ca. 3 min. Pre- and posttreatment samples from eight patients (five chronic and three transplant) who failed lamivudine treatment were amplified, and the presence of mutations in codon 550 was determined by ABI sequencing and by using the LightCycler; in some cases PCR products were also cloned, and multiple clones were sequenced. Concordant results were obtained in all cases. We found the LightCycler to be better at resolving the sequences of genomic mixtures; for example, two samples showed a sequence at codon 550 of (A/G)T(G/T), which was found by fluorimetry to be mixtures of GTG and ATT but no ATG, and this finding was confirmed by the sequencing of clones. However, this approach was not more sensitive than population sequencing for the detection of the presence of mixtures. Overall, this pilot study has demonstrated an approach that could be an extremely rapid and economical method for the detection of lamivudine resistance-associated mutations in HBV.