中国兽医科学2013，Vol.43Issue(3)：261-265,5.
猪嵴病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立
Development of a real-time fluorescent quantitative PCR assay for the detection of porcine kobuvirus
摘要
Abstract
The aim of this study was to develop a one-step real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) assay using a SYBR Green I dye for the detection of porcine kobuvirus in fecal samples. The assay comprised of amplifying a cDNA template generated from viral RNA and establishment of a standard curve using in vitro transcribed porcine kobuvirus RNA. The results showed that the test has a detection limit as low as 100 copies/μL RNA, a reaction efficiency of 91. 8%,and a correlation coefficient (r2) of 0. 992. The fluorogenic RT-PCR assay was determined to be 100-fold more sensitive than conventional RT-PCR. Furthermore,melting curve analysis showed no primer-dimers or non-specific products were produced. The one-step SYBR Green I RT-PCR developed was specific, sensitive, and reproducible for the quantification of porcine kobuvirus in fecal samples. This method may be further optimized for use as a reliable assay for diagnosing and monitoring porcine kobuvirus infections in pigs.关键词
猪嵴病毒/实时荧光定量PCR/定量Key words
porcine kobuvirus/SYBR Green I RT-PCR/quantitation分类
农业科技引用本文复制引用
刘孟良,王潇娣,徐薇薇,周远成,陈雷,朱玲,徐志文..猪嵴病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立[J].中国兽医科学,2013,43(3):261-265,5.基金项目
四川省杰出青年基金项目(200930421) （200930421）
教育部新世纪优秀人才支持计划项目(NCET 11-1059) （NCET 11-1059）
