大鲵蛙病毒LAMP检测方法的建立

Establishment of a loop-mediated isothermal amplification(LAMP)assay for detection of Chinese giant salamander ranavirus(CGSRV)

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。

Follwing the LAMP protocol,a set of four specific primers was designed based on the sequence of the major capsid protein(MCP)gene of Chinese giant salamander ranavirus(CGSRV).Using CGSRV genomic total DNA as a template,the LAMP assay for detection of CGSRV was developed by optimizing its reaction system and conditions.Its detection limit for CGRSV was determined by detecting the fold diluted CGSRV-DNA and compared with conventional and nested PCR.The optimized reaction temperature and time of CGRSV-LAMP were at 62℃and 40 min,respectively.The detection limit of the LAMP assay for the CGSRV-DNA was determined to 5copies/μL,and that of the both conventional and nested PCR was 5×103and 50copies/μL,respectively,suggesting the LAMP method was much more sensitive.Moreover,the assay was not susceptible to cross reaction with other viruses and bacteria,including Lymphocystis disease virus(LCDV),goldfish herpes virus(CyHV-2),marbled sleepy goby iridovirus(MSGIV),infectious spleen and kidney necrosis virus(ISKNV),Edwardsiella tarda,Aeromonas hydrophilaand Aeromonas veronii.The LAMP amplification produces can be visually inspected after addition of ethidium bromide:positive samples turned pink,while negative samples and no template control reactions remained orange.In conclusion,the LAMP assay for GSRV detection is a convenient,rapid,specific,and sensitive method.