Development of RT-PCR method to detect human enteric viruses |
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학술지명 The Korean Society of Virology
저자 장소영,김상종,오성애,이재인,이찬희,김미숙,이규철
발표일 2009-03-27
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Human enteric viruses are the major cause of acute gastroenteritis outbreaks and a rapid definitive diagnosis is important. For this purpose, nucleic acid-based techniques such as RT-PCR have been developed. Although RT-PCR may provide a rapid, specific and sensitive detection, many steps or reactions are required when detecting many viruses. In this study, we developed a rapid and effective method to detect human enteric viruses with minimal number of reactions. We tested one DNA virus (adenovirus: AdV) and seven RNA viruses including poliovirus (PV), coxsackievirus A (CoxA) and B (CoxB), human rotavirus (HRV), hepatitis A virus (HAV), norovirus (NorV), and astrovirus (AstV). If specific primers were used, it would require 15 reactions, 7 RT and 8 PCR. We tested specific primers, enterovirus universal primers and random primers for RT and PCR of RNA viruses and compared for specificity and sensitivity. We found that random 9-mer could be used for RT reaction of RNA viruses except for HRV without sacrificing sensitivity and specificity. HRV cDNA generated by HRV-specific primer and AdV DNA were amplified in a single tube by duplex PCR. The cDNA generated by RT using random 9-mers could be separated to two reaction tubes: one for duplex PCR detecting enteroviruses (PV, CoxA, CoxB) and HAV, one for duplex PCR detecting NorV and AstV, again without losing sensitivity. In conclusion, we were able to detect 8 enteric viruses with 5 reactions, 2 RTs and 3 PCRs. |