白斑综合症病毒实时荧光LAMP检测方法的建立及应用

研究利用ESE-Quant tube scanner检测平台, 建立了一套基于环介导等温扩增技术(Loop-Mediated Isothermal Amplification, LAMP)的实时荧光检测方法, 用于白斑综合征病毒(White Spot Syndrome Virus, WSSV)的检测; 并在此基础上, 与巢式PCR、Real-time PCR和其他已发表的4种LAMP方法在检测灵敏度、实际应用方面进行比较. 结果显示, 研究建立的实时荧光LAMP检测方法在63℃恒温反应30min可检测到最低为105倍稀释的基因组DNA模板, 与Real-time PCR检测方法的灵敏度相当, 高于巢式PCR和其他已发表的4种LAMP方法的检测灵敏度; 而且特异性较好, 与传染性皮下及造血组织坏死病毒等5种常见对虾病原DNA均无交叉反应. 通过构建质粒进一步进行灵敏度测试显示, 本研究建立的实时荧光LAMP检测方法最低检测限度为24个拷贝质粒DNA, 检出时间亦为30min. 通过对66份待检样品的检测结果显示, 实时荧光LAMP检测方法的检出阳性率为7.57%, 准确率为100%, 高于其他WSSV的检测方法. 因此, 研究建立的WSSV实时荧光LAMP检测方法, 操作简单, 反应速度快, 特异性好, 灵敏度高, 成本低廉, 可以直观、实时地观察反应的进行情况, 适合对虾养殖现场及诊断实验室的WSSV快速检测.  

Real-time fluorescence loop mediated isothernal amplification for the rapid detection of white spot syndrome virus

White spot syndrome virus (WSSV) is a highly lethal and contagious viral pathogen of farmed shrimp and it causes substantial economic losses to the shrimp farming industry worldwide. During the last decade, although considerable progress has been made in the molecular characterization of WSSV, there is no effective therapeutic method available to interfere with the unrestrained occurrence and spread of the disease caused by the virus. So the best way to prevent WSSV infection is through pre-screening of WSSV-free broodstock or larvae and routine surveillance. Therefore, our goal is to establish a simple, rapid and accurate diagnostic method for WSSV in field conditions. In this study, a real-time fluorescence loop mediated isothermal amplification (RealAmp) was developed for WSSV diagnosis by using a portable fluorescence reader named ESE-Quant Tube Scanner, and then the RealAmp method was compared with nested PCR, real-time PCR and other 4 established LAMP methods in their sensitivity. The RealAmp method had a detection limit of 105 dilution template of genomic DNA within 30min under isothermal condition at 63℃, which was the same as that of the real-time PCR, but higher than that of nested PCR and other 4 established LAMP methods. Furthermore, the approach had no signal response to other DNA of shrimp pathogens including infectious hypodermal and hematopoietic necrosis virus (IHHNV). Also, sensitivity assay using pMD18-T-VP28 plasmid which contained the target sequence of the RealAmp method showed that the minimum detection limit was 24 copies of the plasmid within 30min. Moreover, 66 clinical samples of Penaeus vannamei were tested and compared by using the RealAmp method, nested PCR, real-time PCR and other 4 established LAMP methods. The result showed that the positive rate was 7.57% using the RealAmp method, which higher than that of nested PCR and other 4 established LAMP methods, and the accuracy was 100% after sequencing check. Overall, these data revealed that the RealAmp method had an equivalent to the real-time PCR method in specificity and sensitivity for WSSV detection, and has great potential as a field usable molecular tool for routine diagnosis of WSSV.