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根据对虾白斑综合征病毒(WSSV)囊膜蛋白VP28的基因序列设计合成4条特异性引物,以pMDT-VP28重组质粒为标准模板,通过优化反应体系和反应条件,建立了WSSV的环介导等温扩增(LAMP)检测方法,测定LAMP对WSSV-DNA的最低检测限,并与巢式PCR进行了比较。结果显示,该LAMP方法的最佳反应温度为65℃,反应时间为60min;LAMP对WSSV的最低检测限为10copies/μL,而巢式PCR为100copies/μL,表明该LAMP方法的敏感性显著高于巢式PCR检测方法。因此,WSSV的LAMP检测方法比PCR更为简便、快速、灵敏,且无需昂贵的变温仪器,更适应水产养殖的现地检测,本研究为WSSV早期感染的快速检测提供了新方法。
Four specific primers were designed and synthesized according to the gene sequence of the capsid protein VP28 of WSSV. The pMDT-VP28 recombinant plasmid was used as a standard template to optimize the reaction system and reaction conditions to establish a loop-mediated isothermal Amplification (LAMP) assay was used to determine the lowest detection limit of LAMP for WSSV-DNA and to compare it with nested PCR. The results showed that the optimum reaction temperature of this LAMP method was 65 ℃ and the reaction time was 60min. The minimum detectable limit of LAMP to WSSV was 10copies / μL, while the nested PCR was 100copies / μL, indicating that the LAMP method was significantly sensitive In nested PCR detection method. Therefore, the LAMP detection method of WSSV is more simple, rapid and sensitive than PCR, and it does not need expensive temperature-changing instruments and is more suitable for the spot detection of aquaculture. This study provides a new method for rapid detection of early WSSV infection.