斑点叉尾鮰病毒实时荧光LAMP检测方法的建立

REAL-TIME FLUORESCENT LOOP MEDIATED ISOTHERMAL AMPLIFICATION FOR DETECTION OF CHANNEL CATFISH VIRUS

斑点叉尾鮰病毒(Channel catfish virus, CCV)属于大DNA病毒,是一种能引起斑点叉尾鮰(Letalurus punetaus)病毒性疾病的重要病原。研究以编码产物为磷酸激酶的CCV ORF77基因为靶标,设计了能识别靶基因上的8个独立区域的6条特异引物,利用基于环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)建立了用于CCV的实时荧光LAMP快速检测方法。结果显示,建立的实时荧光LAMP检测方法在63℃恒温反应45min条件下,反应大约12min后出现明显的峰值,最低检测限度为100个拷贝的病毒质粒DNA,检出时间为35min;并且具有良好的特异性,与白斑综合症病毒(WSSV)、传染性皮下及造血组织坏死病毒(IHHNV)、锦鲤疱疹病毒(KHV)、真鲷虹彩病毒(RSIV)、传染性脾肾坏死病毒(ISKNV)和新加坡石斑鱼虹彩病毒(SGIV)等常见水生动物病原DNA均没有交叉反应;同一样品于试验内及试验间重复性试验发现,20个平行样品的扩增曲线基本重合,表现出良好的重复性。通过对实际样品的检测结果显示,实时荧光LAMP检测方法能够特异性的检出CCV。因此,研究建立的CCV实时荧光LAMP检测方法操作简单、检测快速、特异性好、灵敏度高、结果可靠,能为斑点叉尾鮰养殖现场和实验室的斑点叉尾鮰疱疹病毒病病原的快速诊断和实时监测方面提供技术支撑。

Channel catfish virus(CCV)is a highly lethal viral pathogen of channel catfish(Letalurus punetaus),and it is a large DNA virus that can cause lethal hemorrhagic infection in channel catfish.The establishment of a simple,rapid and accurate CCV detection technique is essential for early diagnosis and timely disease control on site.In this study,a real-time fluorescent loop mediated isothermal amplification(RealAmp)was developed for CCV diagnosis and a set of six primers was designed specifically to recognize the target gene ORF77 coding phosphokinase protein of CCV.The results showed that the RealAmp methods can quickly detect the template of pMD18-T-ORF77 within 12min under isothermal condition of 63℃and the minimum detection limit was 100 copied within 35min.Furthermore,it was found that the method is highly specific without any cross-reactions to other DNA pathogens from aquatic animals including White spot syndrome virus(WSSV),Infectious hypodermal and hematopoietic necrosis virus(IHHNV),Kio herpesvirus(KHV),Red sea bream iridovirus(RSIV),Infectious spleen and kidney necrosis virus(ISKNV)and Singapore grouper iridovirus(SGIV).The intra-assay and inter-assay tests showed that the amplification curves of the parallel samples were substantially coincident,indicating a good reliability.The actual sample results showed that RealAmp method can specifically detect CCV.Overall,these data suggest that the RealAmp method for CCV detection is an effective and low-cost procedure with high specificity and sensitivity,and is expected to become a valuable molecular tool for rapid detection and identification of CCV.