CAJ | 학술논문

以患鲤疱疹病毒2型（Cyprinid herpesvirus 2， CyHV－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、 Hel－ly′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。
이환리포진병독2형（Cyprinid herpesvirus 2， CyHV－2）질병적이육은즉（Carassius auratus gibelio）신장위실험재료，채용－20℃냉동、100％을순、 Dafano′s 액、10％복이마림、 Zenker′s 액、 Müller′s 액、2.5％무이철、 Hel－ly′s 액、 Mossman′s 액、 Bouin′s 액화 Carnoy′s 액불동방법진행보존，통과미량분광광도계검측화경지당응효전영탐토불동보존방법대병독 DNA 제취효과、상규 PCR 확증효과화소식 PCR 확증효과적영향。결과표명：종병어신장중제취 DNA 시，제100％을순보존방법여－20℃냉동보존조일양가이제취고질량적 DNA 외，기타보존방법대 DNA 제취유불동정도적영향，100％을순보존방법화보존적재료가이용작우리포진병독2형 DNA 적제취；상규 PCR 확증시，100％을순、 Zenker′s 액、2.5％무이철、 Helly′s 액、 Bouin′s 액화 Carnoy′s 액보존방법여－20℃냉동보존조구유상동적효과，재362 bp 처출현명량일치적청석단일목적조대，저6충보존방법급기보존적재료가이용작리포진병독2형적상규 PCR 확증；소식 PCR 확증시，100％을순、 Dafano′s 액、10％복이마림、 Zenker′s 액、 Müller′s 액、2.5％무이철、 Helly′s 액、 Mossman′s 액、 Bouin′s 액화 Carnoy′s 액소유보존방법여－20℃냉동보존조구유상동적효과，재339 bp 처출현명량일치적청석단일목적조대，저사보존방법급기보존적재료균가이용작우리포진병독2형적소식 PCR 확증，재채용소식 PCR 진행검측화진단환리포진병독2형질병상구유응용개치。
Kidney of Cyprinid herpesvirus 2 (CyHV-2) diseased Carassius auratus gibelio was used as experimental materi-al and preserved in -20 ℃ as frozen diseased group, 100% ethanol, Dafano′s solution, 10% formalin, Zenker′s solu-tion, Muller′s solution, 2.5% glutaraldehyde, Helly′s solution, Mossman′s solution, Bouin′s solution and Carnoy′s so-lution, respectively.Based on the viral DNA extraction effect , conventional PCR amplification effect and nested PCR am -plification 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% etha-nol can be used for CyHV-2 DNA extraction; Concerning conventional PCR amplification , a single bright 362 bp DNA band from diseased fish kidneys were amplified which were preserved in 100% 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 CyHV -2; Con-cerning nested PCR amplification, a single bright 339 bp DNA band from diseased fish kidneys preserved in all preserva -tive methods of 100% ethanol, Dafano′s solution, 10% formalin, Zenker′s solution, Muller′s solution, 2.5% glutaral-dehyde, 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 amplifica -tion of CyHV-2 and have applicational values in using nested PCR for detecting and diagnosing CyHV -2 disease.