Publication:
Multiplex RT-nested PCR differentiation of gill-associated virus (Australia) from yellow head virus (Thailand) of Penaeus monodon

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Issued Date

2004

Resource Type

Article

File Type

application/pdf

ISSN

1660934

DOI

10.1016/j.jviromet.2003.11.018

Other identifier(s)

2-s2.0-1442335321

Rights Holder(s)

มหาวิทยาลัยศรีนครินทรวิโรฒ

Bibliographic Citation

Journal of Virological Methods. Vol 117, No.1 (2004), p.49-59

Suggested Citation

Cowley J.A., Cadogan L.C., Wongteerasupaya C., Hodgson R.A.J., Boonsaeng V., Walker P.J. Multiplex RT-nested PCR differentiation of gill-associated virus (Australia) from yellow head virus (Thailand) of Penaeus monodon. Journal of Virological Methods. Vol 117, No.1 (2004), p.49-59. doi:10.1016/j.jviromet.2003.11.018 Retrieved from: https://hdl.handle.net/20.500.14740/6441

Title

Multiplex RT-nested PCR differentiation of gill-associated virus (Australia) from yellow head virus (Thailand) of Penaeus monodon

Author(s)

Cowley J.A.
 Cadogan L.C.
 Wongteerasupaya C.
 Hodgson R.A.J.
 Boonsaeng V.
 Walker P.J.

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.

Subject(s)

RNA
 Virus RNA
 Amplicon
 Animal tissue
 Article
 Australia
 Gene amplification
 Gene sequence
 Genetic variability
 Gill associated virus
 Infection
 Lymphoid organ
 Multiplex real time nested polymerase chain reaction
 Nonhuman
 Penaeus monodon
 Priority journal
 Promoter region
 Real time polymerase chain reaction
 Sensitivity analysis
 Sequence analysis
 Shrimp
 Strain difference
 Thailand
 Virus
 Virus detection
 Virus infection
 Virus strain
 Yellow head virus
 Amino Acid Sequence
 Animals
 Australia
 Base Sequence
 Conserved Sequence
 DNA Primers
 Gills
 Molecular Sequence Data
 Nidovirales
 Nucleic Acid Denaturation
 Penaeidae
 Reverse Transcriptase Polymerase Chain Reaction
 RNA, Viral
 Sequence Alignment
 Sequence Homology, Nucleic Acid
 Thailand
 Decapoda (Crustacea)
 Gill-associated virus
 Monodon
 Penaeidae
 Penaeus monodon
 Yellow head virus

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URI

https://hdl.handle.net/20.500.14740/6441

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Scopus: Year 1983-2021

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