不同方法保存的患病异育银鲫中鲤疱疹病毒2型DNA提取和PCR扩增效果分析

Analyses of Cyprinid herpesvirus 2 DNA extraction and PCR amplification from diseased Carassius auratus gibelio preserved by different methods

以患鲤疱疹病毒2型(Cyprinid herpesvirus 2,Cy HV-2)疾病的异育银鲫(Carassius auratus gibelio)肾脏为实验材料,采用-20℃冷冻、100%乙醇、Dafano's液、10%福尔马林、Zenker's液、Müller's液、2.5%戊二醛、Helly's液、Mossman's液、Bouin's液和Carnoy's液不同方法进行保存,通过微量分光光度计检测和琼脂糖凝胶电泳探讨不同保存方法对病毒DNA提取效果、常规PCR扩增效果和巢式PCR扩增效果的影响。结果表明:从病鱼肾脏中提取DNA时,除100%乙醇保存方法与-20℃冷冻保存组一样可以提取高质量的DNA外,其它保存方法对DNA提取有不同程度的影响,100%乙醇保存方法和保存的材料可以用作于鲤疱疹病毒2型DNA的提取;常规PCR扩增时,100%乙醇、Zenker's液、2.5%戊二醛、Helly's液、Bouin's液和Carnoy's液保存方法与-20℃冷冻保存组具有相同的效果,在362 bp处出现明亮一致的清晰单一目的条带,这6种保存方法及其保存的材料可以用作鲤疱疹病毒2型的常规PCR扩增;巢式PCR扩增时,100%乙醇、Dafano's液、10%福尔马林、Zenker's液、Müller's液、2.5%戊二醛、Helly's液、Mossman's液、Bouin's液和Carnoy's液所有保存方法与-20℃冷冻保存组具有相同的效果,在339 bp处出现明亮一致的清晰单一目的条带,这些保存方法及其保存的材料均可以用作于鲤疱疹病毒2型的巢式PCR扩增,在采用巢式PCR进行检测和诊断患鲤疱疹病毒2型疾病上具有应用价值。

Kidney of Cyprinid herpesvirus 2( Cy HV-2) diseased Carassius auratus gibelio was used as experimental material and preserved in- 20 ℃ as frozen diseased group,100% ethanol,Dafano's solution,10% formalin,Zenker's solution,Muller's solution,2. 5% glutaraldehyde,Helly's solution,Mossman's solution,Bouin's solution and Carnoy's solution,respectively. Based on the viral DNA extraction effect,conventional PCR amplification effect and nested PCR amplification effect of three levels by detections of trace spectrophotometer and agarose gel electrophoresis,the influences of different preservative methods were explored. Results showed that high quality of viral DNA could be extracted from the kidneys of diseased fish preserved in 100% ethanol and in- 20 ℃ frozen diseased group. Other preservative methods had different degrees of influences on DNA extraction. Therefore,preservative method and preserved materials of 100% ethanol can be used for Cy HV-2 DNA extraction; Concerning conventional PCR amplification,a single bright 362 bp DNA band from diseased fish kidneys were amplified which were preserved in100% ethanol,Zenker's solution,2. 5% glutaral-dehyde,Helly's solution,Bouin's solution and Carnoy's solution and- 20 ℃ frozen diseased group,respectively. These six preservative methods and their preserved materials can be used for conventional PCR amplification of Cy HV-2; Concerning nested PCR amplification,a single bright 339 bp DNA band from diseased fish kidneys preserved in all preservative methods of 100% ethanol,Dafano's solution,10% formalin,Zenker's solution,Muller's solution,2. 5% glutaraldehyde,Helly's solution,Mossman's solution,Bouin's solution and Carnoy's solution and- 20 ℃ frozen diseased group were obtained,respectively. All preservative methods and their preserved materials can be used for nested PCR amplification of Cy HV-2 and have applicational values in using nested PCR for detecting and diagnosing Cy HV- 2 disease.