The use of differential display to isolate viral genomic sequence for rapid development of PCR-based detection methods - A test case using Taura syndrome virus
The purpose, of this study was to explore the efficacy of using differential display (DD) to isolate viral genomic sequence using tissues from infected organisms so that a PCR procedure to detect the pathogen may be developed rapidly. The model virus used was the Taura syndrome virus (TSV), a ssRNA virus that cause high rates of mortality at shrimp farms. Two random primers in combination with four anchored primers were used to isolate five cDNAs. ranging in size from 241 to 822 bp, that were differentially expressed in TSV-infected shrimp (Litopenaeus vannamei). PCR experiments revealed that four of the five encoded shrimp genes while the fifth was likely to be a TSV gene. Evidence that the putative TSV sequence is part of the TSV genome was obtained by the 97% sequence identity it shared with the published TSV genome. PCR primers were designed successfully using the differential display sequence to develop a RT-PCR-based method to detect TSV. Because differential display does not require physical isolation of the virus and only a small amount of infected sample is needed, the technique may be useful as a method to isolate nucleic acid sequences from emerging pathogens so that PCR primers for their detection may be developed rapidly. (C) 2004 Elsevier B.V. All rights reserved.