One-tube, probe-based, quantitative PCR assay for bidnavirus MHBV in the river prawn Macrobrachium rosenbergii
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Issued Date
2025-01-30
Resource Type
ISSN
00448486
Scopus ID
2-s2.0-85203197460
Journal Title
Aquaculture
Volume
595
Rights Holder(s)
SCOPUS
Bibliographic Citation
Aquaculture Vol.595 (2025)
Suggested Citation
Gangnonngiw W., Bunnontae M., Senapin S., Wongprasert K. One-tube, probe-based, quantitative PCR assay for bidnavirus MHBV in the river prawn Macrobrachium rosenbergii. Aquaculture Vol.595 (2025). doi:10.1016/j.aquaculture.2024.741566 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/102958
Title
One-tube, probe-based, quantitative PCR assay for bidnavirus MHBV in the river prawn Macrobrachium rosenbergii
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Corresponding Author(s)
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Abstract
Macrobrachium hepatopancreatic bidnavirus (MHBV) was first identified in 2023 from the hepatopancreas of moribund river prawns, Macrobrachium rosenbergii (Mr) post-larvae (PL). Developing a rapid and precise quantitative PCR (qPCR) method would enable accurate screening of broodstock river prawns and PL for MHBV. This study developed a specific and sensitive probe-based qPCR assay for the detection of MHBV. Primers and probes were designed from the MHBV genome sequence MN714903 available at GenBank. Two MHBV genes, ORF3 (non-capsid protein) and ORF4 (DNA polymerase domain) were evaluated as target genes. ORF3 exhibited approximately 4-cycles higher sensitivity, thus it was selected for further development. The method developed in this study involved a one tube, duplex qPCR designed to simultaneously target ORF3 from MHBV and an internal housekeeping gene (elongation factor) from prawns. Subsequently, the protocol was validated using templates comprising DNA extracted from Mr, both infected and uninfected with MHBV, and standard DNA plasmids containing the qPCR target sequences. The duplex qPCR method demonstrated high efficiency (92.14 %) with an analytical sensitivity as low as 10 plasmid copies per reaction and a Cq value cut off at 36.40 (± 0.63). Analytical specificity testing was performed using templates extracted from shrimp infected with various viruses (DHPV, XSV, MrNV, WSSV, YHV, PmNV, IHHNV, and IMNV) and all gave negative results. Furthermore, the application of this qPCR method to archived DNA samples extracted from asymptomatic Mr in 2005, 2006, 2010, and 2011 revealed the long presence of MHBV in Thailand before its discovery in 2023 in moribund Mr PL dually infected with MHBV and DHPV. This qPCR method not only provides a valuable tool for the specific detection of MHBV in shrimp but also sheds light on the historical prevalence of MHBV in river prawns.