数字RT-PCR检测鲤春病毒血症的方法建立与应用

Establishment and Application of Digital RT-PCR Assay for Detection of Spring Viraemia of Carp

[目的]通过实时定量RT-PCR和数字RT-PCR两种方法的比对试验,建立快速检测鲤春病毒血症的数字RT-PCR方法。[方法]利用同一对引物和探针,以梯度稀释的方法测定两种方法针对SVCV的灵敏性、特异性及重现性。[结果]两种方法均能够检测出104倍稀释的SVCV,而数字RT-PCR可以检测出单个微滴中的SVCV,其灵敏度性高于实时定量RT-PCR。同时,两种方法的特异性都很强,对其他病毒,如传染性胰腺坏死病毒(IPNV)、传染性造血器官坏死病毒(IHNV)、病毒性出血败血病毒(VHSV)均未有扩增反应。两种方法的重现性也较好。[结论]数字RT-PCR方法比实时定量RT-PCR具有更高的灵敏度,在鲤春病毒血症的早期快速诊断方面具有重要作用。

Objective]Toestablished a rapid digital RT-PCR assay for detection of spring viraemia of carp virus by comparison ofreal-time quantitative RT-PCR and digital RT-PCR. [Methods] Same primers and probes were used in the digital RT-PCR assay and the real-time quantitative RT-PCR to test their sensitivity,repeatability and specificity for detection of SVCV by means of gradient dilution.[Result] Both the digital RT-PCR and real-time quantitative RT-PCR assays could detect SVCV diluted to 10-4,and the former could detect SVCV in a single droplet,more sensitive than the real-time quantitative RT-PCR. At the same time,the specificity of the two methods were strong,there were no amplification reaction with other viruses,such as infectious pancreatic necrosis virus（IPNV）,infectious hematopoi-etic necrosis virus（IHNV）,viral haemorrhagic septicemia virus（VHSV）. There was a better reproducibility of the two methods.[Conclusion] Digital RT-PCR had a better sensitivity than the real-time quantitative RT-PCR,and could play an important role in early and rapid diagnosis of spring viraemia of carp virus.