Original

Development of Peptide Nucleic Acid Mediated
Polymerase Chain Reaction Clamping(PMPC)
|Direct Sequencing Method for Detecting
Lamivudine-Resistant Hepatitis B Virus(HBV) Variants
with High Sensitivity and Specificity

Norio OGATA, MD*1, Takafumi ICHIDA, MD*2,
Yutaka AOYAGI, MD*3 and Isao KITAJIMA, MD*4


Reduced sensitivity of HBV to lamivudine, which causes a viral breakthrough during treatment, is attributed to mutations within the tyrosine-methionine-aspartate-aspartate (YMDD) locus in the reverse transcriptase(rt) domain of HBV polymerase, mainly a methionine(rtM204) substitution. The sensitive detection of such mutations before or early in treatment could assist in optimizing antiviral treatment. For this purpose, we developed peptide nucleic acid(PNA) mediated polymerase chain reaction(PCR) clamping(PMPC) with a PNA probe targeting the YMDD locus. We first tested this method for its sensitivity and specificity in detecting a mutant on HBV DNA standards consisting of serial copy number ratios of a known lamivudine-resistant, mutant clone with rtM204I(ATT) to a wild-type clone with rtM204(ATG). The sensitivity was 0.1 to 0.01% in the coexistence of wild-type clones and the specificity was guaranteed by direct sequencing of the products. We next applied this method to HBV DNA specimens extracted from serum from 4 chronic hepatitis B patients treated with lamivudine. Two of these exhibited a break-through of the HBV mutant with rtM204I(ATT), while the other 2 did not. Before treatment, all 4 patients showed HBV with rtM204I encoded by ATA. During treatment, HBV with the rtM204I(ATT) emerged in the 2 breakthrough patients more than 3 months before the breakthrough, whereas this and other known lamivudine-resistant viruses did not appear in the 2 non-breakthrough patients. Thus, our PMPC-direct sequencing method is highly sensitive and reliable for the early identification of lamivudine-resistant HBV that causes a viral breakthrough.
[Rinsho Byori 51 : 313`319, 2003]

yKey Wordszreverse transcriptaseCYMDD motifCpeptide nucleic acidFPNAChuman immunodeficiency virusFHIVCantiviral drugs

Received February 7, 2003; accepted March 19, 2003
*1,4Department of Clinical and Laboratory Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194
*2,3 Third Department of Internal Medicine, Niigata University Hospital, Niigata 951-8520

E-mail :nrykogt@ms.toyama-mpu.ac.jp