A study on identification method of coxsackie virus A16 and enterovirus 71

Abstract

The simple and rapid identification method of coxsackie virus A16 (CA16) and enterovirus 71 (Ev71), the main cause of hand foot and mouth disease, was described in this report. This method was consists of three steps, those were virus isolation, amplification by RT-PCR, and digestion by restriction enzyme Taq I and EcoT22 I. In 1990, many virus strains were isolated in Vero cell line. But after 1994 the number of isolated viruses in Caco-2 cell line increased instead of isolated in Vero cell line. Concerning to isolation of CA16, in 1998, MRC-5 cell line was also used and it's sensitivity was same as Caco-2 cell line. Cytopathic effects were first observed in MRC-5 cell line among these three lines. RNAs of CA1-10, poliovirus 1-3, echovirus 1-7, 9, 11, 14, 16, 17, 18, 24, 25, 27, 30, Ev71, and isolated viruses were extracted by using QIAamp viral RNA kit (QIAGEN). Then two series of reverse transcription using two down stream primers (E31 and E33) were performed. In PCR, the same upper stream primer (primer 2) was used. CA6, CA16 and Ev71 were the only viruses those were not amplified by RT-PCR using primer 2/E31 but amplified by RT-PCR using primer 2/E33. After PCR, PCR products of isolated viruses using primer 2/E33 were digested by Taq I and Eco T22 I. All of Ev71 products were not digested but all of CA16 products were digested. The band pattern of PCR products (CA16) digested by Taq I were divided into three groups. And Eco T22 I digestion pattern is only one. These results were in accord with Taq I and EcoT22 I digestion sites on sequences of CA16 and Ev71. This method should be useful for the rapid identification of CA16 and Ev71.