口蹄疫病毒解旋酶恒温扩增检测方法的建立

摘要

针对FMDV编码3D蛋白(RNA聚合酶)的保守序列设计特异性引物,利用逆转录解旋酶恒温扩增技术(Reverse Transcription helicase-dependent isothermal amplification,RT-HDA)建立了口蹄疫病毒( foot-and-mouth disease virus,FMDV)快速检测方法。该方法可在65℃下,120 min内实现对口蹄疫病毒保守序列的大量扩增。该方法可检测到0.2 ng的FMDV核酸量,比普通RT-PCR高10倍；具有极高的特异性,除了能对FMDV核酸进行扩增外,对其他临床症状与口蹄疫类似的病毒,如水泡病病毒(SVDV)、猪繁殖与呼吸综合症病毒(PRRSV)、猪瘟病毒(CSFV)、猪细小病毒(PPV)和水泡性口炎病毒( VSV)的核酸,则不能扩增出目的片段。 FMDV的RT-HDA检测方法灵敏度和特异性高,而且不需要昂贵的仪器设备,具有广阔的应用前景。%A rapid detection method of foot-and-mouth disease virus ( FMDV) was established based on reverse transcription helicase-dependent isothermal amplification ( RT-HAD) . Its sensitivity and specificity were assessed and compared with RT-PCR method. The results showed that target fragment of FMDV RNA could be amplified by incubating at 65℃ for 120 minutes using a pair of primers designed based on the conservative sequence of foot-and-mouth disease virus genome. The detection limit of this method was 0. 2 ng of RNA sample which was 10 fold higher than that of RT-PCR. The specificity of this method is also high. We couldn’ t detection swine vesicular disease virus( SVDV)、porcine reproductive and respiratory syndrome vi-rus ( PRRSV)、swine fever virus ( CSFV)、porcine parvo virus ( PPV) and vesicular stomatitis virus ( VSV) by this method with the primers of FMDV. RT-HDA detection method of FMDV not only has high sensitivity and specificity,but also does not require expensive equipment. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.