渔业科学进展Issue(4)：59-65,7.DOI:10.11758/yykxjz.20140409
对虾肝胰腺细小病毒滚环扩增检测方法的建立
Development and Evaluation of Rolling Circle Amplification Assay for the Detection of the Hepatopancreatic Parvovirus
摘要
Abstract
Hepatopancreatic parvovirus (HPV), first isolated fromFenneropenaeus merguiensis, is a single-stranded DNA virus that belongs to the Parvoviridae family. It has a spherical shape with an average diameter of 18-22 nm. HPV infects major aquacultured shrimp species, causes slow growth in penaeid shrimp, and consequently seriously threatens the penaeid shrimp farming. Therefore, rapid and cost-effective detection of HPV should help prevent or control disease outbreaks in penaeid shrimp. In this study we established the rolling circle amplification (RCA) assay for the detection of HPV in penaeid shrimp. We used a conservative sequence of a unique gene of HPV to design a padlock probe based on the genomes of all HPVs, and used an unrelated sequence as the primers of HPV-RCA. Using one-step ligation method, we obtained the best results at 4 pmol/L probe with 40 min ligation followed by 30 min amplification at 62℃. The detection limit of HPV-RCA assay was 105copies/μl. HPV-RCA could detect HPV at the lowest concentration of 2 ng/μl in the hepatopancreas DNA in the shrimp samples, whereas the detection limit of HPV-RCA for PHV was 20 pg/μl. Compared to the conventional PCR, the sensitivity of HPV-RCA was 102 lower. However the HPV-RCA probe exhibited very high specificity to the HPV sequence, and showed no cross-reaction with either shrimp genomic DNA or the most common pathogens of shrimp (including infectious hypodermal and hematopoietic necrosis virus, white spot syndrome virus,Monodon baculovirus disease, andVibrio harveyi). Thus HPV-RCA could be established for the diagnosis of HPV infection under a practically isothermal condition. This is a simplified diagnosis method under farm-based experiment conditions, and thus has great potentials for the detection of HPV in both laboratories and the fields.关键词
对虾/肝胰腺细小病毒/检测/滚环扩增Key words
Penaeid shrimp/HPV/Detection/RCA分类
农业科技引用本文复制引用
王勤涛,张庆利,杨昊霖,刘天齐,刘笋,杨冰,黄倢..对虾肝胰腺细小病毒滚环扩增检测方法的建立[J].渔业科学进展,2014,(4):59-65,7.基金项目
公益性行业(农业)科研专项经费(201103034)、现代农业产业技术体系(CARS-47)和中国水产科学研究院基本科研业务费(2012A05)共同资助。 （农业）
