Taura syndrome of penaeid shrimp: cloning of viral genome fragments and development of specific gene probes.
The ssRNA genome extracted from purified Taura Syndrome Virus (TSV) was transcribed into double-stranded, blunt-ended cDNA and was used to construct cDNA libraries either in pUC 18 or in pBluescript II KS-vectors. Twelve recombinant plasmids chosen after screening of the libraries were subjected to restriction enzyme digestions for determination of size inserts and restriction maps. Two of them, pP15 and pQ1, were selected for probe construction. The inserts, 1500 and 1300 base pairs (bp) respectively, were DIG-11dUTP-labelled and the corresponding probes were named P15 and Q1. On northern blots and dot blots, using different denaturation methods, the 2 probes hybridized specifically with extracted RNA-TSV genome, TSV and infected TS shrimp homogenates. No positive hybridization was obtained with other shrimp viruses tested [Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and Hepatopancreatic Parvovirus (HPV)]. The specificity of the 2 probes was confirmed by in situ hybridization on histological sections of TS diseased shrimps.
Mari
J
J
Department of Veterinary Science, University of Arizona, Tucson 85721, USA.
Bonami
J R
JR
Lightner
D V
DV
D000818:Animals; D003001:Cloning, Molecular; D015342:DNA Probes; D018076:DNA, Complementary / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis*; D033364:Decapoda / Q000821:virology*; D015723:Gene Library; D017403:In Situ Hybridization; D010849:Picornaviridae / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D012680:Sensitivity and Specificity
D015342:DNA Probes; D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral