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.
Lu
Yufeng
Y
Department of Biological Sciences, The University of Southern Mississippi, 118 College Drive 5018, Hattiesburg, MS 39406, USA.
Wang
Shiao Y
SY
Lotz
Jeffrey M
JM
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary; D020869:Gene Expression Profiling; D016679:Genome, Viral; D033561:Penaeidae / Q000821:virology; D012328:RNA Viruses / Q000235:genetics* / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D012367:RNA, Viral