钟亮宁,何洪桂,李小军,叶毅飞,王红兵,刘烺,潘杰,谢海燕,莫钻兰,赖颖,黄洁莹,朱燕秋,李敏,张险朋

• 1:东莞市动物疫病预防控制中心
• 2:东莞市人兽共患病重点实验室
• 3:广东俊一锦鲤养殖有限公司

摘要(Abstract)：

【目的】构建一种敏感性高、特异性强、重复性好的锦鲤疱疹病毒（Koi herpesvirus,KHV）微滴式数字PCR(ddPCR）检测方法，为锦鲤疱疹病毒的定量检测和低浓度样品检测提供技术支持。【方法】参照靶位基因锦鲤疱疹病毒TK基因（GenBank登录号：KX609547.1）设计合成引物和探针，通过比对筛选出最佳引物探针，优化反应体系和退火温度，建立与实时荧光PCR方法的线性关系，并分析该方法的敏感性、特异性、重复性，最后应用于临床样品检测。【结果】当引物、探针浓度分别为900、300 nmol/L且退火温度为57℃时，建立的KHV ddPCR扩增反应效率最高，阴阳性微滴分布界限最为明显。KHV ddPCR敏感性强，检测限低至0.46拷贝/μL，在0～8.1×10~4拷贝/μL范围内，与实时荧光PCR检测结果的线性关系较佳（R~2=0.994 9）。检测变异系数低，批内变异系数为2.70%，批间变异系数为3.02%。与鲤浮肿病毒、鲫造血器官坏死病毒、真鲷虹彩病毒、草鱼出血病毒、罗非鱼湖病毒、十足目虹彩病毒、虾肝肠胞虫（Enterocytozoon hepatopenaei）和诺卡氏菌（Nocardia seriolae）等其他8种常见的水生动物疫病阳性样品无非特异性反应。在88份的临床样品检测中，阳性2份，阳性检出率为2.27%；在8份能力验证样品检测中，5份阳性，与能力验证满意结果一致，与实时荧光PCR方法和套式PCR方法检测结果一致。【结论】建立的锦鲤疱疹病毒ddPCR检测方法敏感性高、特异性强、重复性好，可定量检测锦鲤疱疹病毒DNA，为锦鲤疱疹病毒的研究提供参考。

关键词(KeyWords)： 锦鲤疱疹病毒;微滴式数字PCR;线性关系;特异性;变异系数

Abstract:

Keywords:

基金项目(Foundation): 东莞市2021年省乡村振兴战略专项资金（“大专项+任务清单”）项目（20211800400112）;; 东莞市社会发展科技项目（20231800939872）

作者(Author): 钟亮宁,何洪桂,李小军,叶毅飞,王红兵,刘烺,潘杰,谢海燕,莫钻兰,赖颖,黄洁莹,朱燕秋,李敏,张险朋

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