Use of real-time PCR and fluorimetry to detect lamivudine resistance-associated mutations in hepatitis B virus

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 wi th built-in fluorometric detection. Primers were designed to amplify approx imately 100 bp of the polymerase gene of hepatitis B virus (HBV) spanning c odon 550, where mutations associated with resistance to lamivudine invariab ly occur. Four hybridization probes were synthesized: one was 3' labelled w ith fluorescein and hybridized upstream of codon 550. The others were 5' la belled with Cy5 and 3' labelled with biotin and spanned codon 550. The Cy5- labellcd oligonucleotides contained either wild-type (ATG) or mutant (GTG o r ATT) sequences. A Cy5-labelled probe and either the fluorescein-labelled probe or Sybr Green 1 (a compound that fluoresces when bound to double stra nded 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 c a. 3 min. Pre- and posttreatment samples from eight patients (five chronic and three transplant) who failed lamivudine treatment were amplified, and t he 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 cas es. We found the LightCycler to be better at resolving the sequences of gen omic 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 bu t no ATG, and this finding was confirmed by the sequencing of clones. Howev er, this approach was not more sensitive than population sequencing for the detection of the presence of mixtures. Overall, this pilot study has demon strated an approach that could be an extremely rapid and economical method for the detection of lamivudine resistance-associated mutations In HBV.