摘要
为了建立能特异检测不同基因型猪瘟病毒(Classical swine fever virus,CSFV),同时又能区分其他瘟病毒的基因检测方法,本实验针对CSFV基因组5′端非编码区设计并合成了简并引物和TaqMan探针,在优化反应条件的基础上,成功地建立了特异检测CSFV的荧光定量RT-PCR检测方法。再以已知滴度的CSFV石门株血毒总RNA反转录产物建立标准品,该标准品可以用于定量临床样品中的CSFV滴度,所建立的荧光定量PCR方法可以灵敏地检测出10^(-0.82)个TCID_(50)病毒含量。最后用建立的方法对108份临床样品进行检测并同时进行病毒分离,荧光定量PCR方法检测出73份阳性样品且与病毒分离的符合率为100%,而常规RT-PCR只检测出54份阳性样品,表明本荧光定量RT-PCR法在检测猪瘟病料上具有潜在的应用价值。
In this study, a real-time RT-PCR method was developed to detect and differentiate various genotypes of Classical swine fever virus （CSFV） from other pestiviruses using a set of degenerate primers and a TaqMan probe directed to the 5＇ NTR of classical swine fever virus genome. A serial cDNA standards were prepared by reverse transcription using RNA template prepared from blood stock of CSFV strain Shimen and used to optimize the real-time PCR. The method was successfully used to quantitate the virus load in tissue specimens, and was able to detect 10^-0.82 TCID50 of the virus. Further detection of 108 clinical tissue specimens showed that 73 of them were CSFV positive by the real-time RT-PCR, which were subsequently confirmed by virus isolation after 1 to 3 passages on PK-15 cells. In comparison only 54 of them were detected positive in routine RT-PCR, indicating that real-time RT-PCR is more sensitive than routine RT-PCR, which has a potential application in clinical diagnosis.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2007年第6期467-470,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
科技部重大动物疫病技术平台项目(2004BA519A45)
国家自然科学基金课题(30371071)资助
