LAMP快速检测对虾IHHNV的方法与应用

Development of Rapid Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus by Loop-mediated Isothermal Amplification

建立了一种检测对虾传染性皮下及造血组织坏死病毒(Infectious hypodermal and hematopoietic necrosis vi-rus,I HHNV)快速、灵敏的环介导等温扩增(Loop-mediated isothermal amplification,LAMP)方法。针对I HHNV非结构蛋白基因NS1序列的6个保守区域,利用Pri mer Explorer v4.0软件设计4条引物,建立了I HHNV环介导等温扩增快速检测方法,对反应温度和反应时间等参数进行了优化,并将建立的LAMP检测方法与常规PCR检测进行了比较分析。结果表明,LAMP最适反应在65℃恒温条件60min内完成,凝胶电泳呈现特征性梯型条带;反应体系中添加SYBR Green I荧光染料后,绿色的阳性结果很明显区别于橙色阴性结果。LAMP方法的最低检出限为100拷贝/μL,灵敏度较常规PCR高1000倍。用建立的LAMP方法对临床发病南美白对虾样品进行了检测,结果表明建立的LAMP方法适合于对虾I HHNV的现场快速检测。

Loop-mediated isothermal amplification（LAMP） assay is a novel method of gene amplification with high specificity,sensitivity and rapidity,which can be applied for disease diagnosis in shrimp aquaculture.The method is performed under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target.In the present study,according to the conservative regions of non-structural protein gene NS1,a set of four specific primers were designed,and a rapid detection of IHHNV was established by LAMP assay.The parameters of reaction time and temperature were optimized,and its specificity and sensitivity were assessed.The reactions were carried out at 60℃,62℃,63℃,64℃,65℃,66℃,67℃,68℃ for different time（0 min;15 min;30 min;45 min;60 min;75 min）.A plasmid pMDIHHNV carrying target sequence of LAMP detection was constructed.Ten-fold serially diluted pMDIHHNV（107-100copies/μL） was used as template for LAMP assay to investigate the detection limit.To determine the specificity,LAMP assays were carried out with DNA templates from other pathogens（White spot syndrome virus;WSSV,Taura Syndrome Virus;TSV,Aeromonas.hydrophila,V.alginolyticus,Vibrio.parahaemolytious,Escherichia.coli）.The results showed the optimized LAMP assay for the rapid detection of IHHNV was performed at 65℃ for 60min.The LAMP assay had an unequivocal detection limit of 100 copies/μL,and it was 1,000 times lower than that of PCR.The nucleic acids of other pathogens were not amplified by this LAMP system with the specific primers,which showed a good specificity.The resulting amplicons were detected using visual observation after the addition of SYBR Green I and gel electrophoresis.We investigated the efficacy of UNG（uracil-N-glycosylase） and dUTP in avoiding carry-over contamination in the LAMP assay procedure and explored its effect on the amplification efficiency.Products of LAMP with dUTP adding could be lysed by UNG to avoid LAMP products carry-over contamination effectively.The LAMP assay could be finished within an hour,requiring only a regular laboratory water bath or heat block for reaction and the result could easily be detected using visual observation.Clinically suspected IHHNV-infected shrimp samples were detected by both LAMP and PCR assay,and the result indicated that IHHNV was detected rapidly by LAMP instead of by PCR.