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根据大鲵蛙病毒(Chinese giant salamander ranavirus,CGSRV)主要核衣壳蛋白MCP编码基因的序列设计合成4条特异性引物,以重组pMD19-T-MCP质粒为模板,通过反应条件与反应体系优化,建立CGSRV的环介导等温扩增(LAMP)检测方法,测定LAMP对CGSRV的最低检测限,并与巢式PCR和普通PCR进行比较。结果表明,该LAMP方法最佳反应温度为62℃,反应时间为40min,对CGSRV最低检测限5copies/μL,而巢氏PCR和常规PCR的检测限为50和5×103copies/μL,表明该LAMP方法的灵敏度显著高于巢氏PCR和常规PCR;且与淋巴囊肿病毒、鲫鱼出血病疱疹病毒2型、云斑尖塘鳢虹彩病毒、鳜鱼传染性脾肾坏死病毒、迟钝爱德华菌、嗜水气单胞菌和维氏气单胞菌等均无交叉反应;反应结束后加入荧光染料EtBr可肉眼判定粉色为阳性,而棕色则为阴性。因此,该方法对CGSRV的检测较巢氏PCR和常规PCR更为简便、快速、灵敏、特异,且不需昂贵仪器设备,更适合养殖现场检测,为CGSRV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 sequence of the MCP gene of the major nucleocapsid protein of Chinese giant salamander ranavirus (CGSRV). The recombinant pMD19-T-MCP plasmid was used as a template to optimize the reaction conditions and the reaction system CGSRV ring-mediated isothermal amplification (LAMP) detection method, the detection limit of LAMP CGSRV minimum, and compared with the nested PCR and ordinary PCR. The results showed that the optimum reaction temperature was 62 ℃, the reaction time was 40 min, the limit of detection of CGSRV was 5 copies / μL, while the limit of detection of nest PCR and conventional PCR was 50 and 5 × 103 copies / μL, indicating that the LAMP The sensitivity of the method was significantly higher than that of the nested PCR and conventional PCR. The sensitivity of the method to lymphocystis virus, crucian carp hemorrhagic herpesvirus type 2, macular cloud virus, splenomegaly spleen and kidney necrosis virus, Aeromonas and Vibrio aeruginosa, etc. No cross-reaction; after the reaction was added fluorescent dye EtBr macroscopically judged pink positive, while the brown is negative. Therefore, this method is more convenient, rapid, sensitive and specific to the detection of CGSRV than that of the conventional PCR. It does not need expensive equipment and is more suitable for the on-site detection of CGSRV. It provides a new method for the rapid detection of CGSRV infection.