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同时检测五种病原体,使用单管EvaGreen实时PCR试验,具有不同化温度的实时PCR试验

Haoyu Lou^1,2, Xuan Li¹, Guohao Wang¹

¹State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Qingdao, China.

Applied and environmental microbiology

|August 12, 2025

概括

一种新的五重复实时PCR方法同时检测到五种主要的病原体:白斑综合征病毒 (WSSV),传染性皮下和造血性亡病毒 (IHHNV),十足虹色病毒1 (DIV1),Vibrio parahaemolyticus (V_AHPND) 和EHP. 这种快速诊断工具提高了养殖的生物安全性.

科学领域:

水生动物卫生水生动物卫生
分子诊断学分子诊断学
水产养殖病理学水产养殖病理学

背景情况:

甲类疾病对全球产业构成重大威胁,造成了巨大的经济损失.
关键的病原体包括白斑综合征病毒 (WSSV),传染性皮下和造血性亡病毒 (IHHNV),十足虹色病毒1 (DIV1),急性肝胰腺亡病 (AHPND) 引起的Vibrio (V_AHPND) 和EHP.
有效的疾病管理需要快速,准确和同时检测这些病原体的方法.

研究的目的:

开发一种新的基于EvaGreen的五重复化曲线实时PCR方法.
为了同时检测五种主要的病原体:WSSV,IHHNV,DIV1,V,AHPND和EHP.
为病监测和控制提供高效准确的诊断工具.

主要方法:

开发一个五重复的EvaGreen实时PCR测定.
对反应条件的优化,以便同时放大和检测.
使用来自中国养殖地区的临床样本进行验证,评估诊断灵敏度 (DSe) 和特异性 (DSp).

主要成果:

五重试验显示高特异性,只有目标病原体放大.
敏感性测试检测到所有五种病原体的度低至1.0 × 10¹副本/μL.
对800个临床样本的评估显示,所有测试的病原体的DSe>89.74%和DSp为100%.

结论:

开发的五重复EvaGreen实时PCR方法是准确的,敏感的,特定的和可重复的.
这种方法提供了一种有效的方法,可以同时检测EHP,WSSV,V_AHPND,IHHNV和DIV1.
该测试作为一种有价值的工具,用于早期发现病原体,疾病管理和生物安全在养殖业.

关键词:

绿色的夏娃绿色的夏娃化温度化的温度多重检测检测多重检测类病原体的病原体

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相关概念视频

PCR - Polymerase Chain Reaction

PCR - Polymerase Chain Reaction

Real Time RT-PCR

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...