Classical swine fever virus: a second ring test to evaluate RT-PCR detection methods

Six laboratories participated in a study to compare the sensitivity and spe cificity of RT-PCR tests for the detection of classical swine fever virus ( CSFV). Sets of coded samples were prepared by serial dilution of positive s amples and then distributed to each of the laboratories. One set comprised 25 samples of random primed cDNA, synthesised from viral RNA representative of different pestiviruses. The other set comprised samples of blood and se rum obtained from virus-free or CSFV-infected pigs. Each laboratory tested the samples using PCR/RT-PCR according to a set of standardised protocols t hat specified the exact conditions and requirements for inclusion of contro l samples. Two types of test were evaluated. One amplified a part of the 5' -non coding region of the pestivirus genome by means of a closed, one-tube RT-nested PCR. The other amplified a part of the NS5B gene using non-nested ]RT-PCR. The results of the laboratories were compared with one another, an d with those obtained earlier when similar samples were tested by the same laboratories using non-standardised methods [Paton et al,, Classical swine fever virus: a ring test to evaluate RT-PCR detection methods, Vet. Microbi ol., in press]. Standardisation of the protocols resulted in a more consist ent test sensitivity. Three laboratories avoided significant false positive results. Others that did not, could nevertheless recognise that test speci ficity was inadequate from the results obtained with the control samples. M inimum requirements for the inclusion of adequate controls and periodic pro ficiency testing are proposed. Crown Copyright (C) 2000 Published by Elsevi er Science B.V. All rights reserved.