Establishment and Application of Rapid Detection Technology of Infectious Pancreatic Necrosis Virus by Isothermal Amplification

Abstract

为补充现有传染性胰脏坏死病毒(Infectious pancreatic necrosis virus, IPNV)分子生物学检测手段的不足，本研究以Genbank IPNV基因组中编码A片段多聚蛋白的核苷酸序列为靶序列，针对目标序列的6个区域设计了1对RT-LAMP内引物及1对RT-LAMP外引物。通过优化RT-LAMP方法的退火温度、反应时间等反应条件，同时进行了灵敏度试验及特异性试验，建立了IPNV RT-LAMP检测技术。试验结果为：通过优化各反应条件，结果退火温度为62℃、反应时间为45 min时为IPNV RT-LAMP最佳反应条件；特异性试验显示该方法只与目标病原IPNV有扩增反应，与其他水生动物病原无交叉反应，说明该方法具有良好的特异性；灵敏性试验显示该方法能够检测IPNV RNA的低限为0.01 pg RNA，其检测灵敏度是常规PCR方法的100倍，表明本技术具备较高的灵敏度。试验结果表明，本研究建立的IPNV RT-LAMP技术特异性强、敏感性高、操作简便、反应快速，可通过肉眼判定结果，该方法特别适用于传染性胰脏坏死病的现场及野外检测与诊断，为传染性胰脏坏死病的检测及监测提供了重要的技术手段。In order to supplement the lack of molecular biological detection methods of infectious pancreatic necrosis virus (IPNV), the nucleotide sequence encoding A-fragment polypeptide in GenBank IPNV genome was used as the target sequence in this study. A pair of RT-LAMP (reverse transcription loop-mediated isothermal amplification) inner primers and a pair of RT-LAMP outer primers were designed for six regions of the target sequence. The IPNV RT-LAMP detection technology was estab-lished by optimizing the reaction conditions of RT-LAMP, such as annealing temperature and reac-tion time, and sensitivity test and specificity test were carried out. The test results showed that the best reaction conditions of IPNV RT-LAMP were annealing temperature 62℃ and reaction time 45 min by optimizing the reaction conditions. The specificity test showed that this method only had amplification reaction with IPNV and no cross reaction with other aquatic animal pathogens, which indicated that this method had good specificity. The sensitivity test showed that this method can detect IPNV RNA with a low limit of 0.01 pg RNA, and its detection sensitivity is 100 times higher than that of the conventional PCR method, indicating that this technology has high sensitivity. The results show that the IPNV RT-LAMP technology established in this study has strong specificity, high sensitivity, simple operation and fast response, and can be judged by the naked eye. This method is particularly suitable for the field and field detection and diagnosis of infectious pancreatic necrosis disease, and provides an important technical means for the detection and monitoring of infectious pancreatic necrosis disease.