Detection of hepatopancreatic parvovirus in Thai shrimp Penaeus monodon by in situ hybridization, dot blot hybridizationand PCR amplification

Diseases of Aquatic Organisms

Jurairat Phromjai¹  Boonsirm Withyachumnarnkul¹  Timothy W. Flegel¹  Vichai Boonsaeng¹ 

关键词: Hepatopancreatic parvovirus;    HPV;    Penaeus monodon;    Fecal sample;    PCR;    Dot blot hybridization;    In situ hybridization;   

DOI  :  10.3354/dao051227

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

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) byapproximately 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 theseprimers 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 todetect 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.

【 授权许可】

Unknown