Multiplex Reverse Transcriptase-PCR for Simultaneous Detection of Hepatitis B, C, and E Virus
Multiplex Reverse Transcriptase-PCR for Simultaneous Detection of Hepatitis B, C, and E Virus
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DOI：
10.1016/j.jceh.2015.10.001
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发表时间：
2016-03-01
影响因子：
3
通讯作者：
Kar, Premashis
中科院分区：
文献类型：
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作者：
Garg, Gunjan;Kumar, Deepak;Kar, Premashis
前言：B肝炎病毒（HBV）、丙型肝炎病毒（HCV）和戊型肝炎病毒（HEV）可能单独或同时发生在不同类型的肝病患者中。这些病原体的快速、可靠和成本效益高的筛查是大型流行病学研究所必需的。因此，已计划进行一项研究，以开发一种多重逆转录酶-PCR检测方法，该方法可用于一次筛选最大数量的病原体。方法学：为建立同时检测HBV、HCV和HEV的多重RT-PCR方法，对54例HBV、HCV和HEV血清学阳性或非混合感染患者的血清标本进行了单重PCR/RT-PCR和多重RT-PCR检测。这些引物可以检测这些病毒的大多数基因型。使用B、C和E型肝炎病毒的三对特异性引物的混合物，在一个试管中进行多重RT-PCR以鉴定病毒DNA/RNA。通过单重/多重RT-PCR检测的这些病毒的代表性阳性样品也通过测序随后与参考菌株序列比对来确认。结果：多重PCR对HBV、HCV和HEV的特异性为100%，敏感性分别为89%、87%和74%。RT-multiplex PCR的敏感性和特异性与单重PCR有良好的相关性。结论：该研究表明，多重RT-PCR可以作为一种简单可靠的检测方法，用于同时检测HEV的双重感染，特别是在亚洲国家，作为慢性肝病失代偿的原因。
Introduction: The hepatitis B virus (HBV), HCV, and HEV may occur as singly or concurrently in patients of different kind of liver disease. The rapid, reliable, and cost-effective screening of these pathogens is required for the large epidemiological studies. Therefore, a study has been planned to develop a multiplex Reverse Transcriptase-PCR assay which can be used for the screening of maximum number of pathogens at a time. Methodology: To develop multiplex Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay for simultaneous detection of HBV, HCV, and HEV; the serum samples of 54 patients who were positive either singly or inco-infection with for HBV, HCV, and HEV serologically were screened by uniplex PCR/RT-PCR followed by multiplex RT-PCR for HBV, HCV, and HEV using specific primers. These primers can detect most genotypes of these viruses. Multiplex RT-PCR was done in one tube for the identification of viral DNA/RNA using a mixture of three pairs of specific primers for hepatitis B, C, and E viruses. Representative positive samples of these viruses by uniplex/multiplex RT-PCR were also confirmed by sequencing followed by alignment with reference strains sequence. Results: The specificity of multiplex PCR was 100% with high sensitivity 89%, 87%, and 74% for HBV, HCV, and HEV respectively. The sensitivity and specificity of RT-multiplex PCR demonstrated a good correlation with that of uniplex PCR. Conclusion: The study suggests that multiplex RT-PCR can serve as a simple and reliable assay for rapid, sensitive, and cost-effective method for simultaneous detection of super-infections with HEV particularly in Asian countries as a cause of decompensation of chronic liver disease.