Fast, reliable and low cost user-developed protocol for detection, quantification and genotyping of hepatitis C virus
Fast, reliable and low cost user-developed protocol for detection, quantification and genotyping of hepatitis C virus
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DOI：
10.1016/j.jviromet.2013.11.002
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发表时间：
2014-02-01
影响因子：
3.1
通讯作者：
Plaseska-Karanfilska, Dijana
中科院分区：
文献类型：
--
作者：
Davalieva, Katarina;Kiprijanovska, Sanja;Plaseska-Karanfilska, Dijana
HCV感染的早期检测和基因分型对于疾病管理非常重要。重要的是开发快速和成本效益的半自动化技术，允许准确和可重复的检测，定量和基因分型的HCV。拟议的协议包括实时RT-PCR检测HCV检测/定量和类型特异性单管RT-PCR检测基因分型。两种检测试剂盒均按预期检测基因型1-4。实时分析的检测限为112 IU/ml，基因分型分析的检测限为600 +/- 278 IU/ml（平均值+/- SD）。实时检测试剂盒与AMPDECOR HCV v2.0检测试剂盒之间的一致性为100%。实时检测具有宽的线性动态范围内的检测和定量和良好的重现性与2%和0.75%的变异系数，分别为内部和内部测定。观察到的与AMPENTAL COR HCV Monitor v2.0试剂盒的相关性呈线性，相关系数为0.988。对102份样本进行检测，其诊断特异性和敏感性分别为100%和95%，整个HCV诊断过程在6 h内完成，且污染风险较小。除了快速和具有成本效益外，这种方法是可重复的，并且避免了PCR后的酶促和杂交步骤，同时以高临床灵敏度检测和基因分型HCV。(C)2013爱思唯尔有限公司版权所有。
Early detection and genotyping of HCV infection is important for disease management. It is important to develop fast and cost-effective semi-automated techniques allowing an accurate and reproducible detection, quantification and genotyping of HCV.The proposed protocol includes a real-time RT-PCR assay for HCV detection/quantification and a type-specific one-tube RT-PCR assay for genotyping. Both assays detect genotypes 1-4 as intended. The limit of detection was 112 IU/ml for the real-time assay and 600 +/- 278 IU/ml (mean +/- SD) for the genotyping assay. Concordance between the real-time assay and AMPLICOR HCV v2.0 test was 100%. The real-time assay has wide linear dynamic range of detection and quantification and excellent reproducibility with 2% and 0.75% coefficients of variations, for inter- and intra-assays, respectively. The observed correlation with AMPLICOR HCV Monitor v2.0 kit was linear with the correlation coefficient of 0.988. The diagnostic specificity and sensitivity of the genotyping assay, tested on 102 samples, was 100% and 95%, respectively.The overall procedure of HCV diagnosis is completed within 6 h in a closed system with minor contamination risk. In addition to being fast and cost-effective, this approach is reproducible and avoids post-PCR enzymatic and hybridization steps while detecting and genotyping HCV with high clinical sensitivity. (C) 2013 Elsevier B.V. All rights reserved.