Kiatpathomchai Wansika, Taweetungtragoon Attaporn, Jittivadhana Karnyupha, Wongteerasupaya Chainarong, Boonsaeng Vichai, Flegel T W
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand.
J Virol Methods. 2005 Dec;130(1-2):79-82. doi: 10.1016/j.jviromet.2005.06.006. Epub 2005 Jul 15.
A Thai PCR detection method (WSSV-232) yielding a 232 bp amplicon has been used for detection of white spot syndrome virus (WSSV) since 1996. It targets ORF 91 in the full sequence of the only Thai WSSV isolate at GenBank (AF369029). At the beginning of 2002, some Thai shrimp farmers complained that ponds stocked with WSSV-232 PCR negative post-larvae (PL) later suffered WSSV disease outbreaks. Although these outbreaks may have resulted from horizontal transmission of WSSV after stocking, it was also possible that they resulted from false negative PCR test results due to genetic changes at the PCR-assay target after the first appearance of WSSV in Thailand in 1995. Indeed, recent results have revealed at least 12 WSSV variants in Thailand that can be distinguished based on differences in DNA multiple repeat lengths in ORF 94 (GenBank AF369029). To test for variation in the WSSV-232 target sequence in ORF 91, 20 DNA extracts derived from field samples and representing 9 of the WSSV DNA multiple repeat groups were subjected to PCR amplification and sequencing using primers that generated a 403 bp amplicon covering the target for the WSSV-232 assay. An additional three repeat types were included from archived material. Analysis revealed that the 232 bp target sequence in ORF 91 was unchanged in all of the 12 types tested and that the original WSSV-232 detection system was still valid. Thus, any false negative PCR test results leading to farmer complaints would probably have arisen from small sample sizes and low sensitivity of the single-step PCR assay. If so, false negative results could be reduced by the use of nested PCR assays with larger PL sample sizes. .
自1996年以来，一种可产生232 bp扩增子的泰国PCR检测方法（WSSV - 232）一直用于检测白斑综合征病毒（WSSV）。该方法针对GenBank中唯一的泰国WSSV分离株（AF369029）全序列中的ORF 91。2002年初，一些泰国虾农抱怨说，放养了WSSV - 232 PCR检测呈阴性的后期幼虫（PL）的池塘后来爆发了WSSV疾病。虽然这些疫情可能是放养后WSSV水平传播所致，但也有可能是由于1995年WSSV首次在泰国出现后，PCR检测靶点发生基因变化导致PCR检测结果出现假阴性。事实上，最近的结果显示泰国至少有12种WSSV变体，可根据ORF 94中DNA多重重复长度的差异进行区分（GenBank AF369029）。为了检测ORF 91中WSSV - 232靶点序列的变异情况，使用能产生覆盖WSSV - 232检测靶点的403 bp扩增子的引物，对来自野外样本的20份DNA提取物进行PCR扩增和测序，这些提取物代表了9个WSSV DNA多重重复组。另外从存档材料中选取了三种重复类型。分析表明，在所测试的所有12种类型中，ORF 91中的232 bp靶点序列没有变化，原来的WSSV - 232检测系统仍然有效。因此，任何导致虾农投诉的PCR检测假阴性结果可能是由于样本量小和单步PCR检测灵敏度低所致。如果是这样，通过使用巢式PCR检测和更大的PL样本量，可以减少假阴性结果。
