真鲷虹彩病毒SYBR Green I实时荧光PCR方法的建立

Establishment of Real-Time Fluorescent PCR Method for SYBR Green I of Red Sea Bream Iridovirus

摘要: 真鲷虹彩病毒(Red Sea Bream Iridovirus, RSIV)一直被世界动物卫生组织(Office International Des Epizooties, OIE)列为必报疫病病原，为了精准快速检测该病原，本文选取真鲷虹彩病毒保守基因片段为靶序列，设计并合成SYBR Green I实时荧光PCR引物，将另行扩增获得的特异PCR目的基因片段克隆到pMD18-T载体，得到重组质粒标准品。通过优化反应条件，建立了RSIV SYBR Green I实时荧光PCR检测法，并对该方法进行了灵敏度、特异性和重复性分析。试验结果证明，该反应体系的最佳引物浓度为200 nmol/L；检测限最低可达到0.04 pg/反应，其灵敏度比普通PCR要高出10倍。特异性试验表明，该方法与IHNV、IPNV、SVCV和VHSV这4种水生动物病毒无交叉反应，只与目标病原有特异性扩增。重复性试验显示，该方法具备良好的重复性及稳定性。以上结果显示，研究建立的RSIV的SYBR Green I实时荧光PCR方法具有较高的敏感性、良好的特异性及重复性。适用于水生动物及产品中真鲷虹彩病毒的检测与监测，对加强进出口水产品中RSIV的检验检疫具有十分重要的意义。

Abstract: Red Sea Bream Iridovirus (RSIV) has been listed as a notifiable disease pathogen by Office International Des Epizooties (OIE). In order to accurately and quickly detect the pathogen, the conserved gene fragment of red sea bream iridovirus was selected as the target sequence, and SYBR Green I real-time fluorescent PCR primers were designed and synthesized. The specific PCR target gene fragment amplified separately was cloned into pMD18-T vector to obtain the recombinant plasmid standard. By optimizing the reaction conditions, a real-time fluorescent PCR method for RSIV SYBR Green I was established, and the sensitivity, specificity and repeatability of the method were analyzed. The results showed that the optimum primer concentration of the reaction system was 200 nmol/L; the minimum detection limit can reach 0.04 pg/reaction, and its sensitivity is 10 times higher than that of ordinary PCR. The specificity test showed that the method had no cross reaction with IHNV, IPNV, SVCV and VHSV, and only had specific amplification with the target pathogen. The repeatability test shows that the method has good repeatability and stability. The above results show that the SYBR Green I real-time fluorescent PCR method of RSIV has high sensitivity, good specificity and repeatability. It is applicable to the detection and monitoring of red snapper iridovirus in aquatic animals and products, and is of great significance to strengthen the inspection and quarantine of RSIV in import and export products.