Pagani Elisabetta, Pasquetto Valentina, Cemin Chiara, Rossi Patrizia, Crovatto Marina, Gava Graziella, Felder Martina, Huemer Hartwig P, Larcher Clara
Laboratorio Aziendale di Microbiologia e Virologia, Azienda Sanitaria dell'Alto Adige, Bolzano, Italy.
Infez Med. 2012 Jun;20(2):88-92.
We compared a home-made sequencing system to analyze plasma samples from patients with chronic HBV infection with the commercial TRUGENE(®) HBV Genotyping Assay. A PCR and sequencing protocol based on published primers was applied to detect the viral genotypes as well as the major patterns of point mutations leading to resistance to lamivudine, adefovir and entecavir. For the determination of HBV genotypes the obtained sequences were aligned with a database created within the RIDOM TraceEdit program and publicly available reference sequences. Our results showed perfect correlation with the commercial system, with types D (72%) and A (22%) being the most frequent genotypes. The resistance loci were also reliably detected with mostly combined L180M and M204V/I mutations as the local patterns. M204I mutations were more frequent in genotype D, M204V in genotype A isolates. G173L mutations were not found. The only genotype C isolate tested revealed a different pattern (E263D and I269L). These data speak for the usability of this rapid amplification and sequencing approach for routine genotyping of HBV isolates and simultaneous determination of the drug resistance profile of the dominant viral species.
我们将一种自制的测序系统与用于分析慢性乙型肝炎病毒（HBV）感染患者血浆样本的商业TRUGENE（®）HBV基因分型检测方法进行了比较。应用基于已发表引物的聚合酶链反应（PCR）和测序方案来检测病毒基因型以及导致对拉米夫定、阿德福韦和恩替卡韦耐药的主要点突变模式。为了确定HBV基因型，将获得的序列与在RIDOM TraceEdit程序中创建的数据库以及公开可用的参考序列进行比对。我们的结果显示与商业系统具有完美的相关性，其中D型（72%）和A型（22%）是最常见的基因型。耐药位点也能可靠地检测到，主要的局部模式为L180M和M204V/I联合突变。M204I突变在D基因型中更常见，M204V在A基因型分离株中更常见。未发现G173L突变。唯一检测的C基因型分离株显示出不同的模式（E263D和I269L）。这些数据表明这种快速扩增和测序方法可用于HBV分离株的常规基因分型以及同时确定优势病毒株的耐药谱。
