Publication: Detection of hepatopancreatic parvovirus in Thai shrimp Penaeus monodon by in situ hybridization, dot blot hybridization and PCR amplification
Issued Date
2002-10-04
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01775103
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2-s2.0-0037020458
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Mahidol University
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SCOPUS
Bibliographic Citation
Diseases of Aquatic Organisms. Vol.51, No.3 (2002), 227-232
Suggested Citation
Jurairat Phromjai, Vichai Boonsaeng, Boonsirm Withyachumnarnkul, Timothy W. Flegel Detection of hepatopancreatic parvovirus in Thai shrimp Penaeus monodon by in situ hybridization, dot blot hybridization and PCR amplification. Diseases of Aquatic Organisms. Vol.51, No.3 (2002), 227-232. doi:10.3354/dao051227 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/19969
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Title
Detection of hepatopancreatic parvovirus in Thai shrimp Penaeus monodon by in situ hybridization, dot blot hybridization and PCR amplification
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Abstract
Hepatopancreatic parvovirus (HPV) infects the hepatopancreas in penaeid shrimp and retards their growth. The DNA sequence of HPV from Thai shrimp Penaeus monodon (HPVmon) differs from HPV of Penaeus chinensis (HPVchin) by approximately 30%. In spite of this difference, commercial PCR primers (DiagXotics) developed from HPVchin to yield a 350 bp PCR product do give a 732 bp product with HPVmon DNA template. On the other hand, the sensitivity of HPVmon detection with these primers and with hybridization probes designed for HPVchin is significantly lower than it is with HPVchin. To improve sensitivity for HPVmon detection, we used the sequence of the 732 bp HPVmon PCR amplicon described above to develop specific PCR primers (H441F and H441R) and hybridization probe. The primers could detect as little as 1 fg of purified HPVmon DNA while the 441 bp digoxygenin-labeled PCR product gave strong, specific reactions with in situ hybridization and with hybridization blots. In contrast, negative results were obtained using DNA from all other pathogens tested and from DNA of P. monodon. Supernatant solution from boiled, fresh shrimp fecal and postlarval samples homogenized in 0.025% NaOH/0.0125% SDS could be used to detect as little as 0.1 pg HPVmon DNA by the PCR reaction. By dot blot hybridization, a visible signal was obtained with purified HPVmon DNA at 0.01 pg, but detection in spiked feces and postlarval samples was only 1 and 0.1 pg, respectively.