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Diseases of Aquatic Organisms

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DAO 129:145-158 (2018)  -  DOI: https://doi.org/10.3354/dao03243

Real-time PCR tests to specifically detect IHHNV lineages and an IHHNV EVE integrated in the genome of Penaeus monodon

Jeff A. Cowley1,*, Min Rao1, Greg J. Coman2

1Aquaculture Program, CSIRO Agriculture & Food, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, QLD 4067, Australia
2Aquaculture Program, CSIRO Agriculture & Food, Bribie Island Research Centre, 144 North Street, Woorim, QLD 4507, Australia
*Corresponding author:

ABSTRACT: Infectious hypodermal and hematopoietic necrosis virus (IHHNV) can cause mass mortalities in western blue shrimp Penaeus stylirostris, runt deformity syndrome in Pacific white shrimp P. vannamei and scalloped abdominal shell deformities in black tiger shrimp P. monodon. In P. monodon, however, PCR-based diagnosis of IHHNV can be complicated by the presence of a chromosome-integrated, non-replicating endogenous viral element (EVE). To facilitate high-throughput screening of P. monodon for IHHNV infection and/or EVE sequences, here we report real-time PCR tests designed to specifically detect IHHNV Lineage I, II and III but not EVE Type A sequences and vice versa. Using 108 dsDNA copies of plasmid (p)DNA controls containing either IHHNV or EVE-Type A sequences, both tests displayed absolute specificity. The IHHNV-q309 PCR reliably detected down to ≤10 copies of pDNA, at which levels a 309F/R PCR amplicon was just detectable, and the presence of an IHHNV-EVE sequence did not significantly impact its sensitivity. The IHHNV-qEVE PCR was similarly sensitive. Testing of batches of P. monodon clinical samples from Vietnam/Malaysia and Australia identified good diagnostic concordance between the IHHNV-q309 and 309F/R PCR tests. As expected for a sequence integrated into host chromosomal DNA, IHHNV-qEVE PCR Ct values were highly uniform among samples from shrimp in which an EVE was present. The highly specific and sensitive IHHNV-q309 and IHHNV-qEVE real-time PCR tests described here should prove useful for selecting broodstock free of IHHNV infection and in maintaining breeding populations of P. monodon specific pathogen free for IHHNV, and if desired, also free of IHHNV-EVE sequences.


KEY WORDS: Giant tiger shrimp · Prawn · IHHNV · Infectious hypodermal and haematopoietic necrosis virus · Black tiger shrimp · Penaeus stylirostris pestyldensovirus · PstDNV · EVE · Endogenous viral element


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Cite this article as: Cowley JA, Rao M, Coman GJ (2018) Real-time PCR tests to specifically detect IHHNV lineages and an IHHNV EVE integrated in the genome of Penaeus monodon. Dis Aquat Org 129:145-158. https://doi.org/10.3354/dao03243

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