Consensus RT-nested PCR detection of yellow head complex genotypes in penaeid shrimp

Abstract

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 ≤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 ∼1250 and ∼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.