Wijegoonawardane Priyanjalie K M, Cowley Jeff A, Walker Peter J
CSIRO Livestock Industries, Queensland Bioscience Precinct, St. Lucia, Brisbane, Queensland 4067, Australia.
J Virol Methods. 2008 Nov;153(2):168-75. doi: 10.1016/j.jviromet.2008.07.009. Epub 2008 Sep 5.
A consensus RT-nested (n)PCR is described that detects the six distinct genotypic variants in the yellow head virus (YHV) complex. The PCR primers targeted ORF1b gene regions more highly conserved amongst the reference strains of YHV (genotype 1) and gill-associated virus (GAV, genotype 2) and a set of 57 field isolates containing multiple representatives of each genotype. The test employed short PCR (359 bp) and nPCR (147 bp) amplicons to minimise the effects of RNA degradation. To ensure < or = 8-primer degeneracy, two primers were designed to each site, one accommodating sequence variations amongst genotype 1 isolates and the other variations amongst isolates of the other genotypes. The analytical sensitivity limits of the PCR and nPCR were estimated to be approximately 1250 and approximately 1.25 RNA copies, respectively. The superior group-specificity of the consensus RT-nPCR compared to other OIE-recommended PCR tests for YHV/GAV was demonstrated using RNA from 17 Penaeus monodon shrimp infected with representatives of each of the six genotypes. Phylogenetic analysis using the 94 nt ORF1b gene sequence spanned by the nPCR primers generated genotype assignments that were consistent with those obtained using the extended 671 nt sequence used for the initial identification of genotypes.
本文描述了一种共识逆转录巢式（n）PCR方法，该方法可检测黄头病毒（YHV）复合体中的六种不同基因型变体。PCR引物靶向YHV（基因型1）和鳃相关病毒（GAV，基因型2）参考菌株以及一组包含每种基因型多个代表的57个野外分离株中ORF1b基因区域保守性更高的区域。该检测采用短PCR（359 bp）和nPCR（147 bp）扩增子，以尽量减少RNA降解的影响。为确保引物简并度≤8，每个位点设计了两条引物，一条适应基因型1分离株中的序列变异，另一条适应其他基因型分离株中的变异。PCR和nPCR的分析灵敏度极限估计分别约为1250个和约1.25个RNA拷贝。使用感染六种基因型代表的17只斑节对虾的RNA，证明了共识RT-nPCR与其他OIE推荐的YHV/GAV PCR检测相比具有更高的组特异性。使用nPCR引物跨越的94 nt ORF1b基因序列进行系统发育分析，生成的基因型分类与使用用于基因型初始鉴定的671 nt扩展序列获得的分类一致。
