国家质量监督检验检疫总局科研项目（2009IK002，2007IK267）资助
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建立了牙鲆弹状病毒的环介导等温扩增（loop-mediated isothermal amplification，LAMP）检测方法，首先根据牙鲆弹状病毒的糖蛋白的基因保守序列，利用Primer Explorer V3软件设计了6条引物，并对LAMP反应温度和反应时间等条件进行了优化。该方法的检测限为30 fg RNA，比常规RT-PCR灵敏度高100倍，与鲤春血症病毒、传染性胰脏坏死病毒、传染性造血器官坏死病毒、海洋双 RNA 病毒、病毒性出血败血症病毒以及病毒性神经坏死病毒等没有交叉反应。该方法检测时间短，在1 h内即可完成检测。用建立的LAMP方法对临床鱼样进行了检测，结果表明40尾石鲽鱼样品中有3尾感染HRV，与病毒分离结果一致，说明LAMP方法比较适合牙鲆弹状病毒的早期及现场诊断。
A set of six specific primers were designed based on glycoprotein gene of hirame rhabdovirus(HRV) using Primer Explorer V3 software.The parameter of reaction time and temperature was optimized.The primers were specific with HRV and could not amplify spring viraemia of carp virus(SVCV),infectious pancreatic necrosis virus(IPNV),infectious haematopoietic necrosis virus(IHNV),marine birnavirus(MABV),viral haemorrhagic septicemia virus(VHSV) or viral nervous necrosis virus(VNNV).The detection limit of LAMP assay was approximately 30 fg HRV RNA,which was 100-fold higher than that of conventional RT-PCR.The LAMP reaction could be finished within an hour and clinical fish samples were detected using LAMP.The results showed that 3 fish out of 40 samples were infected with HRV by LAMP assay,which was consistent with virus isolation method.The LAMP assay has great potential in field diagnosis of early HRV infection.
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孙颖杰,岳志芹,刘 荭,赵玉然,史秀杰,梁成珠,吴 斌,李 叶,王 哲.牙鲆弹状病毒环介导等温扩增检测方法的建立与应用[J].华中农业大学学报,2010,29(2):203-207
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- 收稿日期:2010-01-04
- 最后修改日期:2010-02-28
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