基于重组酶介导扩增技术诊断鸭瘟方法的建立

Rapid Identification of Duck Plague Based on Recombinase Aid Amplification Assay

以具有种属特异性的鸭瘟病毒gC基因为靶基因,设计引物和荧光标记探针,使用重组酶介导扩增方法(Recombinase aid amplification assay,RAA),39℃恒温扩增,建立了检测鸭瘟病毒的普通和荧光RAA方法。该方法具有良好特异性,仅对鸭瘟病毒检测结果为阳性,而与番鸭细小病毒、小鹅瘟病毒、禽传染性喉气管炎病毒、马立克氏病毒、鸭源沙门菌、鸭源大肠杆菌O157无交叉反应。普通和荧光RAA方法的灵敏度分别为100 copies/μL和10 copies/μL,对28份留存的鸭瘟阳性组织样本和84份疑似鸭瘟临床样品检测结果表明建立的方法与商业化PCR试剂盒的符合率为100%。结果表明,RAA方法实现常温扩增、操作简单、费用低廉、无需热循环过程,是一项具有广泛前景的方法。

According to the species specific gC gene of duck plauge virus,a general and fluorescence recombinase aided amplification assay were developed at temperature 39 ℃.The assay was able to accurately detect duck plauge virus and no cross reactivity with Muscovy duckling parvo virus,gosling parvovirus,avian infectious laryngotracheitis virus,Marek′s disease virus,Salmonella and Escherichia coli 0157 from duck.The sensitivity of this methord was up to100 copies/μL for general RAA and 10 copies/μL for fluorescence RAA,respectively.28 positive archieved samples and84 clinical samples were detected by RAA and commercial PCR kit.The results were 100% consistent with commercial PCR.Compared with classic PCR,the advantages of isothermal amplification at 39 ℃ or under room temperature,simple operation and speediness make RAA a promising molecular detection tool for duck plauge.