使用RT-Seminested PCR方法直接由檢體偵測並鑑定北台灣腸病毒

Abstract

Human enterovirus (HEV) is a genus of the Picornaviridae including more than 87 serotypes belonging to four species designed Human enterovirus A to D. The diagnosis of enterovirus infections, and in particular the characterization of enterovirus strains, relies on the isolation of virus in cell culture from clinical samples. Based cell culture method, there were 24 serotypes of human enteroviruses reported in Taiwan, 2000-June, 2008. Because many enteroviruses do not grow readily in cell culture and that was limited to the low sensitivity, make us unable to study enterovirus prevalence in Taiwan completely. Hence, diagnostic tests based on PCR are developed and found that it is more sensitive and much faster than conventional culture method. This study has planned to use the method-VP1 RT-snPCR (CODEHOP) that developed by Nix and his coworkers in 2006 to detect and identify enteroviruses in northern Taiwan. The specific aims of study are: (1) To detect and identify HEV that could not be isolated by virus culture; (2) To analysis the association between disease and HEV infection; (3) To identify possible new serotypes that haven’t been reported in Taiwan; (4) To identify possible new serotypes that haven’t been reported internationally. 177 virus-culture negative specimens were collected and tested by VP1 RT-snPCR (CODEHOP) and 5’ NTR RT-nPCR methods. VP1 RT-snPCR (CODEHOP) method (67.2%) was more sensitive than 5’ NTR RT-nPCR method (47.9%). Also, VP1 RT-snPCR (CODEHOP) method was more sensitive than virus culture and could increase the positive rate of EV detection. Among 119 CODEHOP-positive samples, 55 EV were identified as CVA24 variant, CVA6, CVA16, and CVA10 mainly by partial VP1 sequence analysis. A new EV serotype candidate was found. The identity of complete VP1 gene of this candidate was less than 70% when compared with reference strains. In addition, there were still 64 EV-positive results that sequence signal were unable to interpret. Therefore, a new method, called CODEHOP HEV-A, B, C screening method was designed to solve those problems. The result show that 39 of 64 specimens were detected by this new method and all positive results could be directly grouped. Among 39 positive samples, there were 10 positive results show mix infection. Moreover the sequence signals were all good to interpret. Although CODEHOP HEV-A, B, C screening method was less sensitive than VP1 RT-snPCR (CODEHOP), it could identify the specimens with two or three serotypes of enteroviruses mix infection. Serotype, CVA24 variant, usually causes acute hemorrhagic conjunctivitis and was never reported association with neural diseases. In this study, there were many cases identified as CVA24 variant through the detection of virus-culture negative specimens. Particularly, CVA24 variants were isolated from the CSF specimens of patients diagnosed with encephalitis and aseptic meningitis. It suggested that CVA24 variant was concerned as the pathogen of neural diseases.