Comparative evaluation of INNO-LiPA HBV assay, direct DNA sequencing and subtractive PCR-RFLP for genotyping of clinical HBV isolates.
Comparative evaluation of INNO-LiPA HBV assay, direct DNA sequencing and subtractive PCR-RFLP for genotyping of clinical HBV isolates.
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DOI：
10.1186/1743-422x-7-111
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发表时间：
2010-05-30
期刊：
影响因子：
4.8
通讯作者：
Al-Nakib W
中科院分区：
文献类型：
--
作者：
Ali MM;Hasan F;Ahmad S;Al-Nakib W
乙型肝炎病毒 (HBV) 的基因型（A 至 H）影响 HBV 感染患者的肝病进展和抗病毒治疗反应。已经开发出多种方法用于 HBV 菌株的快速基因分型。然而，其中一些方法可能不适合发展中国家。通过分析慢性 HBV 患者的血清 (n = 80) 样本，评估了 INNO-LiPA HBV 基因分型测定 (LiPA)、直接 DNA 测序和基因型特异性 HBV 基因组区域的消减 PCR-RFLP 的性能，以准确确定 HBV 基因型。 LiPA 和 DNA 测序均鉴定出 63 株、4 株和 13 株 HBV 菌株分别属于 D 基因型、A 基因型以及 A 和 D 混合基因型。相反，基于 PCR-RFLP 的方法正确鉴定了所有 4 种 A 基因型菌株，但仅正确鉴定了 63 种 D 基因型菌株中的 56 种。七种 D 基因型菌株产生了不确定的结果。 DNA 序列比较表明，目标区域中的单核苷酸变化产生了 Nla IV 的额外限制位点，从而损害了该方法的准确性。此外，所有混合基因型A和D菌株仅被鉴定为基因型A菌株。数据显示，基于PCR-RFLP的方法错误地识别了一些D基因型菌株，并且未能识别混合基因型感染，而LiPA和DNA测序则产生了准确的结果。
Genotypes (A to H) of hepatitis B virus (HBV) influence liver disease progression and response to antiviral therapy in HBV-infected patients. Several methods have been developed for rapid genotyping of HBV strains. However, some of these methods may not be suitable for developing countries. The performance of INNO-LiPA HBV Genotyping assay (LiPA), direct DNA sequencing and subtractive PCR-RFLP of genotype-specific HBV genome regions were evaluated for accurately determining the HBV genotypes by analyzing sera (n = 80) samples from chronic HBV patients. Both, LiPA and DNA sequencing identified 63, 4 and 13 HBV strains as belonging to genotype D, genotype A and mixed genotype A and D, respectively. On the contrary, the PCR-RFLP-based method correctly identified all 4 genotype A but only 56 of 63 genotype D strains. Seven genotype D strains yielded indeterminate results. DNA sequence comparisons showed that a single nucleotide change in the target region generated an additional restriction site for Nla IV that compromised the accuracy of this method. Furthermore, all the mixed genotype A and D strains were identified only as genotype A strains. The data show that the PCR-RFLP-based method incorrectly identified some genotype D strains and failed to identify mixed genotype infections while LiPA and DNA sequencing yielded accurate results.