检测施马伦贝格病毒核酸的荧光定量RT-PCR方法

A Real-time Quantitative Reverse Transcription-PCR for the Detection of Schmallenberg Virus Nucleic Acid

根据施马伦贝格病毒(Schmallenberg virus,SBV)S基因节段设计一对引物和TaqMan探针,建立了检测SBV核酸的荧光定量RT-PCR方法。通过优化反应体系和反应条件,该方法对101~107半数组织培养感染量(TCID50)的SBV核酸呈现良好的线性关系。利用德国FLI制备和保存的SBV核酸阳性(n=140)、SBV核酸阴性(n=132)和辛波血清群相关病毒核酸样品(n=16),对该方法的敏感性、特异性和重复性进行了验证。结果表明,所建立方法与FLI研制的SBV荧光定量RT-PCR具有相近的敏感性,两者对140份SBV核酸阳性样品的检测符合率达96.4%(135/140);可灵敏地检测出1TCID50的SBV RNA,并具有良好的重复性;除道格拉斯病毒(Douglas virus)外,与辛波血清群相关病毒核酸无交叉反应。用该方法对采自内蒙古呼和浩特市某养殖场的绵羊血清(n=47)和澳大利亚进口绵羊血清(n=47)的RNA进行了检测,结果未发现SBV核酸阳性样品。SBV荧光定量RTPCR检测方法的建立为应对施马伦贝格病提供了有效的检测手段。

In the present study,apair of primers and a TaqManprobe were designed based on the S-segment sequence of Schmallenberg virus(SBV),and a real-time quantitative reverse transcription-PCR(RT-qPCR)for the detection of SBV nucleic acid was established.After optimization of the reaction system and condition,the method presented a good linear relationship with the RNA template ranging from 101 to 107 50%tissue culture infectious dose(TCID50)SBV.By using SBV nucleic acid-positive samples(n=140),SBV nucleic acid-negative samples(n=132),and RNA samples(n=16)of related viruses within the Simbu serogroup,the sensitivity,specificity and reproducibility of the RT-qPCR were validated.All the samples used for validation were prepared and preserved by the Friedrich-Loeffler-Institut(FLI)of Germany.The results demonstrated that the developed RT-qPCR had a similar sensitivity with that of FLI and the concordance rate between the two assays was 96.4%(135/140),that the RT-qPCR could sensitively detect 1TCID50 SBV RNA and it had a good reproducibility,and that the RT-qPCR showed no cross-reactivity with related viruses within the Simbu serogroup except for Douglas virus.The RT-qPCR was further used to detect the RNA samples extracted from sheep sera which were collected from a breeding farm located in Inner Mongolia(n=47)and from the sheep imported from Australia(n=47),respectively.However,no SBV nucleic acid-positive sample was detected.The successful development of SBV RT-qPCR provides us with an efficient detection tool for dealing with Schmallenberg disease.