新型竹鼠源阿卡斑病毒RT-PCR检测方法的建立

Establishment of RT-PCR Method for Detection of Novel Akabane Virus Isolated from Bamboo Rats

为了建立新型竹鼠源阿卡斑病毒RT-PCR的检测方法,通过对新型竹鼠源阿卡斑病毒(Akabane virus,AKAV)及其同属各病毒基因序列进行比对分析,设计了针对AKAV M基因片段的一对特异性引物,经优化建立AVKV的RT-PCR检测方法。结果表明,在57℃退火温度条件下,于3h内可完成目的片段扩增,目的片段大小为816bp;该方法能特异性地扩增新型竹鼠源AKAV,不能扩增竹鼠细小病毒、竹鼠圆环病毒、竹鼠内源性反转录病毒、竹鼠乳酸脱氢酶增高病毒;检验敏感性高,灵敏度可达23pg。对感染发病竹鼠样品的检测结果显示,本方法特异准确,能清楚分辨发病竹鼠不同组织中病毒核酸丰度。说明建立的RT-PCR方法简单特异、灵敏度高,不仅适用于实验室鉴定,还可应用于口岸、野外等的临床监测。

To establishment of the RT-PCR method for detection of the novel Akabane virus isolated from bamboo rats,based on sequence analysis of the novel Akabane virus isolated from bamboo rats,the RT-PCR primers were designed targeting the M gene of AKAV,and the RT-PCR assay was established after optimization and the assay was finished within3hours at57℃annealing temperature.The results showed that this detection method had high specificity and it can amplify the M gene of Akabane virus,no detection appeared with bamboo rat parvovirus,bamboo rat circovirus,bamboo rat endogenous retrovirus and lactate dehydrogenase-elevating virus.The detection was high sensitive and it limit was23pg.Test results of intracerebrally inoculated bamboo rat samples showed that the method was accurate,and the virus abundances of the different organ samples were clearly identified.The established RT-PCR was simple,specific and sensitive assay,suitable for use both in laboratory test and clinical field surveillance.