A non-stop, single-tube, semi-nested PCR technique for grading the severity of white spot syndrome virus infections in Penaeus monodon

Diseases of Aquatic Organisms

Sarawut Jitrapakdee¹  Chainarong Wongteerasupaya¹  Sakol Panyim¹  Wansika Kiatpathomchai¹  Anchalee Tassanakajon¹  Vichai Boonsaeng¹ 

关键词: White spot syndrome virus;    WSSV;    Single-tube nested PCR;    Penaeus monodon;   

DOI  :  10.3354/dao047235

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

ABSTRACT: A single-tube, non-stop, semi-nested polymerase chain reaction (PCR) technique was developed for simultaneous detection and severity grading of white spot syndrome virus (WSSV) infections in the black tiger shrimp Penaeus monodon. Thetest uses 1 sense primer and 3 anti-sense primers that produce up to 3 PCR products (1100, 526 and 250 base pairs [bp]) depending upon the severity of infection. Specifically, heavy infections (≥2 × 10⁴ viralparticles) of WSSV produce all 3 fragments, while moderate infections (around 2 × 10³ viral particles) produce 2 (526 and 250 bp) and light infections (20 to 200 viral particles) produce 1 (250 bp). In addition, thetechnique uses internal control primers that yield a shrimp characteristic fragment for non-infected samples and samples with a low quantity of viral target in order to assure integrity and reproducibility of the PCR assays. The non-stop, single-tube,semi-nested PCR technique is simple and convenient and can detect as little as 5 fg WSSV DNA (20 viral particles) in crude extracts of postlarval samples or extracts of pleopods and haemolymph from larger shrimp.

【 授权许可】

Unknown