猪细环病毒2型环介导等温扩增检测方法的建立

Development of Loop-mediated Isothermal Amplification Assay for Detection of Torque teno sus Virus Type 2

为了能快速、特异的检测猪细环病毒2型(TTSuV2),本研究针对TTSuV2全基因序列的非编码区域和第1个开放性阅读框前端设计了2对引物,建立了TTSuV2的环介导等温扩增(LAMP)检测方法并对反应成分和条件进行了梯度摸索。试验结果显示,该LAMP检测方法最佳反应条件为64℃恒温90 min,可特异性检测TTSuV2,与猪细环病毒1型、猪圆环病毒2型、猪瘟病毒、猪繁殖与呼吸综合征病毒和猪博卡病毒无交叉反应,病毒最低检出限为100拷贝/μL。结果表明,建立的LAMP方法具有快速、特异且灵敏的特点,可在TTSuV2快速检测方面提供一定的技术支持。

In order to develop a loop-mediated isothermal amplification （LAMP） assay for rapid and specific detection of Torque teno sus virus type 2 （TTSuV2）,two pairs of primers were de- signed according to the untranslated regions and the part of open reading frame 1 of TTSuV2. The LAMP system was optimized by adjusting the concentrations of some components and reaction conditions. The optimized amplification conditions of LAMP assay was at 64 ℃ for 90 min. The results showed the LAMP assay was specific for TTSuV2 detection,which could achieve a detec- tion limit of 100 copies/μL viral nucleic acid, and no cross-reaction with TTSuV1, PCV2,CSFV, PRRSV and PBoV. In conclusion, this assay was a rapid, specific and sensitive detection technique which could provide a assistance for the rapid detection of TTSuV2.