非洲马瘟病毒TaqMan探针荧光定量RT-PCR检测方法的建立

Development of real-time fluorescent quantitative RT-PCR assay for detection of African horse sickness virus using TaqMan probes

为建立非洲马瘟病毒(AHSV)TaqMan探针荧光定量RT-PCR检测方法,根据GenBank及自己所测的AHSV 9个血清型VP7的ORF全长核苷酸序列,设计2套位于AHSV基因组S7片段的特异引物及相应TaqMan探针。经试验,结果证实2套引物探针可将9种血清型的AHSV RNA分为2群而分别进行特异性检测,而对与AHSV同属的蓝舌病病毒、鹿出血热病毒及正常马淋巴组织均无荧光信号可检出。其中探针H2及其配套引物可特异性检测AHSV1、3、4、6、7、8、9七个血清型,而MGB探针及其配套引物可特异性检测AHSV 2、5两个血清型。分别以构建好的2型、9型全长VP7的pMD18-T-VP7质粒为标准品建立标准曲线,最终建立了AHSV TaqMan荧光探针实时荧光定量RT-PCR快速检测方法,可实现对全部9个血清型AHSV的定量检测和快速诊断。

In order to establish the method of real-time fluorescent quantitative RT-PCR assay for de- tection of African horse sickness virus（AHSV） using TaqMan probes,two pairs of specific primers and two probes all located on the segment 7 were designed, based on the VP7-ORF sequences retrieved from Gen- Bank and of the sequences of nine serotypes of AHSV previously completed by our own team. The results showed that the nine serotypes of AHSV could be classified into two groups by using the two primer pairs. Specific fluorescent signal could be detected when each of the primer pairs and its related probe were used, but no specific signal in those genetically related viruses,including blue tongue virus,epizootic haemorrha- gic disease virus and normal lymphoid tissue of horse. Seven serotypes of AHSV including serotypes 1,3, 4,6,7,8 and 9 could be specifically detected with the probe H2 and its paired primers, and the other two se- rotypes 2 and 5 could be specifically identified with the MGB probe. Positive recombinant plasmids （AHSV2-pMD18-T-VP7 and AHSV9-pMD18-T-VP7） were constructed and used for positive quantitative template to establish standard curves, respectively. Finally, the method of real-time fluorescent quantitative RT-PCR assay for detection all of AHSV serotypes based on TaqMan probe was established and could be used for rapid diagnosis and quantitative detection.