Please use this identifier to cite or link to this item:
Title: Multiplex RT-nested PCR differentiation of gill-associated virus (Australia) from yellow head virus (Thailand) of Penaeus monodon
Authors: Cowley J.A.
Cadogan L.C.
Wongteerasupaya C.
Hodgson R.A.J.
Boonsaeng V.
Walker P.J.
Keywords: RNA
virus RNA
animal tissue
gene amplification
gene sequence
genetic variability
gill associated virus
lymphoid organ
multiplex real time nested polymerase chain reaction
Penaeus monodon
priority journal
promoter region
real time polymerase chain reaction
sensitivity analysis
sequence analysis
strain difference
virus detection
virus infection
virus strain
yellow head virus
Amino Acid Sequence
Base Sequence
Conserved Sequence
DNA Primers
Molecular Sequence Data
Nucleic Acid Denaturation
Reverse Transcriptase Polymerase Chain Reaction
RNA, Viral
Sequence Alignment
Sequence Homology, Nucleic Acid
Decapoda (Crustacea)
Gill-associated virus
Penaeus monodon
Yellow head virus
Issue Date: 2004
Abstract: A multiplex RT-nested PCR has been developed to detect and differentiate the closely related prawn viruses, gill-associated virus (GAV) from Australia and yellow head virus (YHV) from Thailand. RT-PCR using primers to conserved sequences in the ORF1b gene amplified a 794bp region of either GAV or YHV. Nested PCR using a conserved sense primer and either a GAV- or YHV-specific antisense primer to a divergent sequence differentially amplified a 277bp region of the primary PCR amplicon. Multiplexing the YHV antisense primer with a GAV antisense primer to another divergent sequence allowed the viruses to be distinguished in a single nested PCR. Nested PCR enhanced detection sensitivity between 100- and 1000-fold and GAV or YHV RNA was detectable in ∼10fg lymphoid organ total RNA. The multiplex RT-nested PCR was also able to co-detect GAV and YHV RNA mixed over a wide range of concentrations to simulate potential dual-infection states. The robustness of the test was examined using RNA samples from Penaeus monodon prawns infected either chronically or acutely with GAV or YHV and collected at different locations in Eastern Australia and Thailand between 1994 and 1998. GAV- (406bp) or YHV-specific (277bp) amplicons were differentially generated in all cases, including five YHV RNA samples in which no primary RT-PCR amplicon was detected. Sequence analysis of GAV and YHV PCR amplicons identified minor variations in the regions targeted by the virus-specific antisense primers. However, none occurred at positions that critically affected the PCR. Crown Copyright © 2003 Published by Elsevier B.V. All rights reserved.
ISSN: 1660934
Appears in Collections:Scopus 1983-2021

Files in This Item:
There are no files associated with this item.

Items in SWU repository are protected by copyright, with all rights reserved, unless otherwise indicated.