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

根据对虾肝胰腺细小病毒(HPV)保守基因序列,设计特异性的锁式探针及其扩增引物,优化反应条件,建立了肝胰腺细小病毒超分支滚环扩增检测方法。实验中采用一步法连接,探针在Taq DNA连接酶作用下,58℃连接40 min、62℃扩增30 min便可以扩增出明显条带。反应特异性验证实验表明,该体系能够特异性地检测出HPV,而不与供试的其他对虾病原发生交叉反应;灵敏度分析结果显示该方法的检测极限为105 copies/μl,与PCR检测方法相比,一步法连接的滚环扩增的灵敏度低两个数量级。该方法反应过程中温度变化次数少,基本都在等温条件下进行,不需要PCR仪,可发展成为在简便实验条件下使用的简易检测方法。

Hepatopancreatic parvovirus（HPV）, first isolated from Fenneropenaeus 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 10^5 copies/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 10^2 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, and Vibrio 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.