凡纳滨对虾传染性皮下及造血组织坏死病毒(IHHNV)可视化环介导等温扩增(LAMP)技术方法的优化与应用

    Establishment and application of visual loop-mediated isothermal amplification(LAMP) for infectious hypodermal and hematopoietic necrosis virus(IHHNV)

    • 摘要: 传染性皮下及造血组织坏死病毒(Infectious hypodermal and hematopoietic necrosis virus,IHHNV)作为对虾疾病的主要病原之一,能够感染多种对虾,对幼虾危害尤其明显,虽然死亡率不高,但可以引起对虾生长缓慢,造成巨大的经济损失,严重影响对虾养殖业持续健康发展。本文根据IHHNV病毒基因的保守序列,采用Primer Explorer V4软件设计6条LAMP特异性引物组合,建立了一种以环介导等温核酸扩增技术(Loop-mediated isothermal amplification,LAMP)为基础的快速检测IHHNV的方法。对本研究的LAMP检测方法的敏感性和特异性进行分析,并将其灵敏度与实时荧光定量PCR、普通PCR检测方法进行比较。结果显示:LAMP检测方法在63℃恒温条件60 min内完成反应,阳性结果出现可视化的绿色,阴性结果颜色不发生变化;LAMP方法的最低检出限为10.3 copies/μL,灵敏度与荧光实时定量PCR相当,较常规PCR高。结果表明建立的LAMP方法适用于对虾IHHNV的现场快速检测。

       

      Abstract: Infectious hypodermal and hematopoietic necrosis virus(IHHNV), as one of the main pathogens of shrimp diseases, can infect many kinds of shrimp, especially juvenile shrimp. Although the mortality rate of the disease is not high, it can cause slow growth of shrimp, resulting in huge economic losses, and seriously affect the sustainable and healthy development of shrimp farming industry. Six LAMP specific primers were designed by Primer Exploer V4 software according to the conservative sequence of IHHNV gene. And then, we had established a rapid detection method for IHHNV which was based on loop-mediated isothermal amplification(LAMP). The sensitivity and specificity of LAMP detection method in this study were analyzed and compared with quantitative real-time PCR(qRT-PCR) and PCR. The results showed that the LAMP method could complete the reaction within 60 minutes under the constant temperature of 63℃, and the positive results were visualized green, while the negative results did not change in color. The minimum detection limit of LAMP method was 10.3 copies/μL. The sensitivity of LAMP method was similar to that of qRT-PCR, which was higher than that of PCR. 50 samples of Penaeus vannamei collected from different farms were detected by LAMP and PCR respectively. The results of the two methods were consistent. The results showed that the LAMP method was suitable for rapid detection of IHHNV in shrimp.

       

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