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

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.

FIG. 1

Design of fluorescent probes for mutation detection in the LightCycler. HBV consensus sequence in positive sense is shown in boldface. Differences from the consensus for the various genotypes are shown above the consensus sequence, and probes are shown below. The nucleotides encoding position 550 in the probes are underlined. PCRs each contained one Cy5-labelled probe and either the fluorescein-labelled probe or Sybr Green 1. The Cy5 molecule fluoresces only when adjacent to the fluorescein molecule or Sybr Green 1. The probes are designed so that the Cy5 label will detach at a lower temperature than the fluorescein-labelled probe.

FIG. 2

Accumulation of Cy5-specific fluorescence during PCR of serum sample extracts by using WT Cy5 probe together with Sybr Green 1. Viral loads of sera were 14 × 10⁶ genome copies/ml (extract 1), 3.5 × 10⁶ copies/ml (extract 2), 2.9 × 10⁶ copies/ml (extract 3), 0.2 × 10⁶ copies/ml (extract 4), and <400 copies/ml (extract 5).

FIG. 3

Melting peaks of Cy5-labelled probes with two samples from patient A. Sample 1 is an lamivudine-sensitive virus, while sample 2 is a resistant virus. Panels: a, traces obtained with WT probe; b, traces obtained with M1 probe; c, traces obtained with M2 probe. Each trace shows the rate of change of fluorescence with respect to temperature, thus allowing calculation of the temperature at which the probes detach from the PCR product.

FIG. 3

Melting peaks of Cy5-labelled probes with two samples from patient A. Sample 1 is an lamivudine-sensitive virus, while sample 2 is a resistant virus. Panels: a, traces obtained with WT probe; b, traces obtained with M1 probe; c, traces obtained with M2 probe. Each trace shows the rate of change of fluorescence with respect to temperature, thus allowing calculation of the temperature at which the probes detach from the PCR product.

FIG. 3

Melting peaks of Cy5-labelled probes with two samples from patient A. Sample 1 is an lamivudine-sensitive virus, while sample 2 is a resistant virus. Panels: a, traces obtained with WT probe; b, traces obtained with M1 probe; c, traces obtained with M2 probe. Each trace shows the rate of change of fluorescence with respect to temperature, thus allowing calculation of the temperature at which the probes detach from the PCR product.

FIG. 4

Melting peaks of WT probe for five consecutive samples, labelled 1 to 5, obtained from patient A over a period of 14 months. It can be seen that samples 1 to 3 all had a higher melting temperature with this probe than samples 4 and 5.

FIG. 5

Melting peaks of three samples, labelled 1 to 3, from patient C, that were probed with WT and M2 probes. These peaks show the transition with time from ATG to ATG/ATT to ATT. Panels: a, trace obtained with WT probe; b, trace obtained with M2 probe.

FIG. 6

Melting peaks of two samples, labelled 1 and 2, of resistant virus obtained 6 months apart from patient B probed with WT (a), M1 (b), and M2 (c) probes. The peaks show that a mixture of mutant sequences is present.

FIG. 6

Melting peaks of two samples, labelled 1 and 2, of resistant virus obtained 6 months apart from patient B probed with WT (a), M1 (b), and M2 (c) probes. The peaks show that a mixture of mutant sequences is present.

FIG. 6

Melting peaks of two samples, labelled 1 and 2, of resistant virus obtained 6 months apart from patient B probed with WT (a), M1 (b), and M2 (c) probes. The peaks show that a mixture of mutant sequences is present.