UNIVERSAL DIAGNOSTIC RT-PCR PROTOCOL FOR ARBOVIRUSES

A. selected number of PCR protocols were evaluated to determine if the y could serve as a universal protocol for detecting and identifying al l arboviruses. In this study, four parameters that affect the efficacy of RT-PCR (RNA extraction method, choice of reverse transcriptase, ch oice of DNA polymerase and thermocycling program) were evaluated in co mbination. The most optimal combination of those parameters employed u se of silica gel membrane spin column, RAV-2 reverse transcriptase, Tt h DNA polymerase, and a simple modification of a published thermocycli ng program. By this modified protocol, viral RNA could be amplified sa tisfactorily with more than 50 pairs of primers designed for diagnosis of arboviruses representing five families. The sensitivity and specif icity obtained by this universal protocol were comparable to those obt ained by the original protocol for each primer pair tested; and for so me primers, improved sensitivity was observed. It was also found that a simple modification of a suggested protocol of a commercial RT-PCR k it could produce nearly identical results and serve as another univers al protocol. With the use of a universal diagnostic reverse transcript ase-polymerase chain reaction (RT-PCR) protocol, simultaneous screenin g of clinical or biological specimens against a large number of RNA vi ruses belonging to many families can be performed more efficiently for etiologic determination in the situations complicated by the difficul ty of differential diagnosis. Furthermore, such a universal protocol f acilitates reducing the cost of PCR-based diagnostic operation and sta ndardizing the qualities of PCR-based diagnosis within an institution or among collaborating institutions. A logical strategy is to conduct diagnosis in two stages by using broadly group-reactive primers in the first stage to narrow the range of possible etiologic agents and usin g virus-specific primers in the second stage for identification. Befor e such a strategy is employed, however, more group-reactive primers fo r a large number of arboviruses, for which no such primers currently e xist, must be made available. Furthermore, the best pair or pairs of p rimers need to be selected for each virus for the second stage of the strategy. (C) 1998 Published by Elsevier Science B.V. All rights reser ved.