摘要
A simple and rapid assay for the detection of Classical swine fever virus(CSFV)was established using reverse transcription loop-mediated isothermal amplification(RT-LAMP).This study describes the amplification of the genomic RNA of CSFV under isothermal conditions(63℃)within one hour,using a set of six primers(two outer primers,two inner primers and two loop primers).This RT-LAMP assay showed 100-fold higher sensitivity than the standard RT-PCR method and identified eighteen additional positive cases that were negative when tested by RT-PCR.This RT-LAMP was able to detect all the 13 strains of CSFV but not the BVDV.PRRSV.SIV. PRV-PCV,thus showed a good specificity.Products amplified by RT-LAMP can be visualized by agarose gel electrophoresis and in addition,either as a white precipitate at the bottom of the tube after a pulse spin or as a color change when dyed with SYBR Green I which are visible to the naked eye.Because RT-LAMP is low-cost and produces rapid results,it has the potential to be an excellent tool for CSFV surveillance in the field,especially in developing countries.
A simple and rapid assay for the detection of Classical swine fever virus （CSFV） was established using reverse transcription loop-mediated isothermal amplification （RT-LAMP）. This study describes the amplification of the genomic RNA of CSFV under isothermal conditions （63 ℃） within one hour, using a set of six primers （two outer primers, two inner primers and two loop primers）. This RT-LAMP assay showed 100-fold higher sensitivity than the standard RT-PCR method and identified eighteen additional positive cases that were negative when tested by RT-PCR. This RT-LAMP was able to detect all the 13 strains of CSFV but not the BVDV. PRRSV. SIV PRV-PCV, thus showed a good specificity. Products amplified by RT-LAMP can be visualized by agarose gel electrophoresis and in addition, either as a white precipitate at the bottom of the tube after a pulse spin or as a color change when dyed with SYBR Green I which are visible to the naked eye. Because RT-LAMP is low-cost and produces rapid results, it has the potential to be an excellent tool for CSFV surveillance in the field, especially in developing countries
基金
The National Science and Technology supporting plan of the Eleventh Five-year(2006BAD06A18 and 2006BAD06A03)
Beijing Natural Science Foundation(5072041)
关键词
猪瘟病毒
病毒检测
逆转录
琼脂糖凝胶电泳
灯
牛病毒性腹泻
循环使用
投资工具
Classical swine fever virus （CSFV）
Reverse transcription loop-mediated isothermal amplification（RT-LAMP）
Rapid detection
