实时荧光定量PCR法检测对虾白斑综合征病毒

Real-Time Fluorescence Quantitative PCR in Detecting White Spot Syndrome Virus in Penaeid Shrimp

对虾白斑病由白斑综合征病毒（WSSV）感染引起,所有养殖对虾对该病高度易感,蟹类、小龙虾、淡水虾以及各种龙虾也易感,但发病率和死亡率有很大差别。多种虾类常表现为持续感染或终身隐性感染。隐性感染虾体内的病毒含量极低,需建立一种快速、灵敏、准确的检测方法。本文应用TaqMan探针技术设计了探针和引物,建立了检测WSSV的实时荧光定量PCR方法。对影响PCR反应的主要因素Mg^2＋浓度和退火温度等进行了优化,证明当Mg2＋浓度为3.5～4.0 mmol,退火温度为59～60℃时可获得最佳的扩增和检测效果。

White spot disease（WSD） is considered to be infection with white spot syndrome virus（WSSV）.All farmed penaeid shrimp species are highly susceptible to infection,often resulting in high mortality.Crabs,crayfish,freshwater prawns,spiny lobsters and clawed lobsters are susceptible to infection,but morbidity and mortality as a consequence of infection is highly variable.Persistent infection occurs commonly and lifelong infection has been shown.Disease outbreaks may be induced by stressors,such as rapid changes in salinity.Viral loads during persistent infection can be extremely low and potentially undetectable by any available diagnostic test.It is important to establish a quick,sensitive and veracious detection method in the prevention of the disease.The TaqMan probe which was the most common technique in real-time fluorescence quantitative PCR was used in this report to examine WSSV.The different concentration of Mg^2＋ and the anneal temperature of PCR which affected the reaction mostly had been tested.It was proved that the optimal reaction system was 3.5 to 4.0 mmol of Mg2＋ and 59 to 60℃ for anneal temperature.