基因1型狂犬病病毒一步法荧光定量RT-PCR检测方法的建立

目的在我国狂犬病均由基因1型狂犬病病毒（rabies virus,RV）引起。本研究针对RV N基因保守序列设计并合成了一套简并引物和Taqman探针,在优化反应条件的基础上,建立了检测RV核酸的一步法荧光定量RT-PCR（qRT-PCR）检测方法。方法与结果该方法能特异检测基因1型,不能检测基因2-7型和犬瘟热病毒（CDV）、犬细小病毒（CPV）、犬腺病病毒（CAV）、水泡性口炎病毒（VSV）、日本乙型脑炎病毒（JEV）等5种非狂犬病病原体,其检测灵敏度可达到4.68个TCID50的病毒含量。用该方法对29份新鲜和5份腐败的临床犬脑组织样品进行检测,并与国际金标准确诊方法狂犬病荧光抗体染色法（FAT）和乳鼠脑内接种试验（MIT）及本实验室建立的套式RT-PCR方法进行比较。结果表明所建立的qRT-PCR与套式RT-PCR的符合率为100%,二者均检测出12份阳性的新鲜样品和5份阳性的腐败样品,而FAT只检测出12份阳性的新鲜样品和1份阳性的腐败样品,MIT只检测出12份阳性,未检测出阳性腐败样品。检测中FAT和MIT确诊的所有阳性样品在qRT-PCR检测均是阳性,而在FAT检测为阴性的4份腐败样品在qRT-PCR为阳性,说明所建立的qRT-PCR方法准确性达到了FAT和MIT的水平,而且灵敏度更高,更适合于腐败样品的检测。结论研究结果表明该qRT-PCR方法特异性好、灵敏度高、污染率低、操作简单,在我国动物狂犬病临床诊断上具有巨大的应用价值。

Establishment of the one-step real-time RT-PCR assay for detection of genotype 1 lyssavirus

In present study,the one-step real-time RT-PCR（qRT-PCR） was developed to detect the rabies virus using a set of degenerate primers and TaqMan probe directed to the leader sequences and 3′ ends of N gene.The specificity assay showed that the method could only detect the classical rabies virus,not the other six lyssaviruses from genotype 2 to 7,and other viruses such as CDV,CPV,CAV,VSV and JEV.The detection limit of the established method was 4.68 TCID50.To validate the method,29 fresh and 5 decomposed dog brain specimens submitted to our laboratory for rabies confirmation were detected in comparison with OIE golden diagnostic standard methods FAT and MIT and routinely-used nested RT-PCR.In the result,the real-time RT-PCR and nested RT-PCR yield 12/29 and 5/5 positive detection respectively for fresh and decomposed samples,showing 100% similarity,while FAT was positive in 12/29 fresh and 1/5 decomposed samples,and MIT positive in 12/29 fresh and 0/5 decomposed samples.All positive samples tested by FAT and MIT were also positive in the real-time RT-PCR,but the later was more sensitive than FAT and MIT and particularly useful for decomposed brain tissue.This study shows that the established real-time RT-PCR has good specificity,sensitivity,low cross-contamination and easier operation,which rendering the method is very applicable in clinical diagnosis of classical rabies.