白斑综合症杆状病毒(WSBV)PCR检测方法的改进及应用

IMPROVEMENT AND APPLICATION OF AN IMPROVED PCR METHOD FOR DETECTING WHITE SPOT SYNDROME BACULOVIRUS (WSBV) OF PENAEUS MONODON

斑节对虾Penaeusmonodon白斑综合症是由白斑综合症杆状病毒 (WSBV)所致。患有典型白斑综合症的斑节对虾 ,用两种提取病毒模板DNA的方法 ,进行PCR扩增 :一种是用传统的方法 ,即蛋白酶K ,CTAB等消化 ,酚 /氯仿抽提 ,乙醇沉淀得到DNA ;另一种是用改进的煮沸法提取病毒DNA。对比发现后一种方法稳定性及敏感性比前者高 ,重复性好。用后一种方法对病虾的复眼进行检测 ,PCR结果为阳性。应用后一种方法还对病虾的各组织器官及人工感染的虾进行检测 ,发现病虾中除肝胰腺外 ,附肢、肌肉、心脏、鳃、胃、中肠、神经、复眼、表皮结果均为阳性。感染实验中 ,出现阳性结果注射感染比投喂感染早 ,而且肌肉和附肢较敏感。

Two methods of extracting WSBV DNA were used for PCR amplification. One is traditional, with shrimp specimens of white spot syndrome digested in proteinase K and CTAB, WSBV DNA extracted by phonl chloroform and precipitated with absolute ethanol. The other is an improved method, with shrimp specimens of white spot syndrome boiled and then directly used for PCR amplification of WSBV DNA. Comparing the two methods, the improved one was more stable and sensitive than the traditional one. It was found that the detecting results of the pereiopods, muscle, gills, heart, stomach, midgut, nervous tissue, integument as well as compound eyes and eye stalks from the diseased shrimps were all positive by the improved method. It was also found that muscle and pereiopods were more sensitive than other tissues or organs.