Paton D J, McGoldrick A, Bensaude E, Belak S, Mittelholzer C, Koenen F, Vanderhallen H, Greiser-Wilke I, Scheibner H, Stadejek T, Hofmann M, Thuer B
Department of Virology, Veterinary Laboratories Agency, Central Veterinary Laboratory - Weybridge, Woodham Lane, New Haw, Surrey KT15 3NB, Addlestone, UK.
Vet Microbiol. 2000 Nov 15;77(1-2):71-81. doi: 10.1016/s0378-1135(00)00264-9.
Six laboratories participated in a study to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Sets of coded samples were prepared by serial dilution of positive samples 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 serum obtained from virus-free or CSFV-infected pigs. Each laboratory tested the samples using PCR/RT-PCR according to a set of standardised protocols that specified the exact conditions and requirements for inclusion of control 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, and 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. Microbiol., in press]. Standardisation of the protocols resulted in a more consistent test sensitivity. Three laboratories avoided significant false positive results. Others that did not, could nevertheless recognise that test specificity was inadequate from the results obtained with the control samples. Minimum requirements for the inclusion of adequate controls and periodic proficiency testing are proposed.
六个实验室参与了一项研究，以比较用于检测经典猪瘟病毒（CSFV）的逆转录聚合酶链反应（RT-PCR）检测方法的敏感性和特异性。通过对阳性样本进行系列稀释制备编码样本集，然后分发给每个实验室。一组包括25个随机引物cDNA样本，这些样本由代表不同瘟病毒的病毒RNA合成。另一组包括从无病毒或感染CSFV的猪获得的血液和血清样本。每个实验室根据一套标准化方案使用PCR/RT-PCR检测样本，该方案规定了纳入对照样本的确切条件和要求。评估了两种检测类型。一种通过封闭的单管逆转录巢式PCR扩增瘟病毒基因组5'-非编码区的一部分。另一种使用非巢式RT-PCR扩增NS5B基因的一部分。将各实验室的结果相互比较，并与这些实验室早期使用非标准化方法检测类似样本时获得的结果进行比较[帕顿等人，经典猪瘟病毒：评估RT-PCR检测方法的环式试验，《兽医微生物学》，即将发表]。方案的标准化导致检测敏感性更加一致。三个实验室避免了显著的假阳性结果。其他未避免假阳性结果的实验室，仍可从对照样本的检测结果中认识到检测特异性不足。提出了纳入适当对照和定期能力验证的最低要求。
