Establishment of a Real-Time PCR Analysis System to detect Enterovirus infections
2002 Vol.18 NO.11
There have been several enteroviris outbreaks in Taiwan since the summer of 1998. Though these outbreaks have been controlled, there have been some serious infections of children and even deaths. Two laboratory methods are traditionally used to confirm diagnosis, the cell culture infectivity(1), and a molecular biological diagnosis method such as PCR(2). A new method, real-time PCR, has been developed to directly detect enterovirus in specimens. This method combines the Tag-Man technique and the ABI PrismTM 7900 real-time sequencing detection system. The method is highly sensitive, specific, as well as time and manpower-saving. The designed primers are specific for the non-coding region of the 5’ section (a highly conserved region). One-step RT-PCR(3) was carried out to amplify to about 145 bps sections. By using the dual fluorescent labeled DNA probe and the AmpliTaq DNA’s 5’→3’ nucleolytic activity, specific PCR products can be detected and compared with standard of known quantity to estimate indirectly the quantity of the virus. To more precisely quantify the amount of enterovirus in specimens, the present study used Mahoney type 1 polivirus RNA as standard and molecular cloning of the amplified PCR products from polivirus RNA into the pGEM-T Easy Vector. The dynamic range of this assay encompassed at least 7 orders of magnitude (101 - 107). It is useful in the mass screening of specimens. Any enteroviral titer within 101 - 107 in specimens can be detected immediately after real-time PCR reaction; the conventional way of detecting the presence of PCR products after PCR reaction is not necessary.