Detection Of Classical Swine Fever Virus By A Real-Time RT-PCR

Classical swine fever (CSF) is a highly contagious viral disease ofswine.CSF is a significant impediment to global trade in products derivedfrom swine and very substantial economic losses result from outbreaks indomestic swine.It is one hundred and seventies years since the firstoutbreak of CSF which is still prevalence in many countryworldwide,such as parts countriesand region in South America, Asia,Europe. Other countries such as Franch,Netherland,Genman, Belgiumoccurrenced CSF again when they had proclaimed the eliminating ofCSF.Due to carraying out inoculation of HCLV vaccine,the large scaleoutbreaks in China is shutted down despite of continuous appearencr insmall scale.Why do this would happen? With the change of epidemicmodel and clinical manifestation of CSF,new born piglets and beingweaned pigs often is infected,while species pig and bearing pig often haveinsisitence.The clinical manifestation transformed from acute model tothe coexistence of acute model and chronicity atypical form which ischaracteristiced by long pathogenesis,low incidence and mortality. Thisproblem results from immune failure and low ability of immunity.Theatypical CSF and immune failure,together with mixing infection withother infectious diseases of swine,all this have made the diagnosing ofCSF very difficult,which request new diagnostic technique for CSF. It was important of diagnosis CSF rapidly and exactly,which relatedwith the development of international trade and the balance ofeconomy.There is still a long distance between the diagnostic techniquefor CSF and the need of practice. Rapid,specific and precise diagnosticmethods of CSFV is emergency request.In order to cope with the new change of epidemiology and supplying thecurrent diagnostic methods,we estabilished a Real Time PCR motheds of detectionof CSFV by detection of viral nucleic acid.The Real Time PCR motheds elevate thelevel of detection CSF and would be used as a foundation tool in CSFV research.In this study a set of degenerate primers and a TaqMan probedirected to 5'NTR of Classical Swine Fever Virus genome weresynthesized and used to establish a real-time RT-PCR method to detectand discriminate various genotype of classical swine fever virus fromother pestiviruses. Furthermore the reverse transcription(RT) productmade from blood stock of CSFV strain Shimen with known titer was usedto generate serial viral RNA standards in real-time RT-PCR. Aftersystematic optimization the method was successfully adapted toquantitate the virus load in tissue specimens and able to detect as lessamounts as 10-3.97 TCID50 of the virus. Further detection of 108 clinicaltissue specimens by real-time RT-PCR in comparison with virus isolationand routine RT-PCR showed that 79 of them were CSFV positive inreal-time RT-PCR as well as in virus isolation after 1 to 3 passage onPK-15 cells, while only 54 of them the virus positive in routine RE-PCR,indicating that real-time RT-PCR is faster and more sensitive than routineRT-PCR and virus isolation and has a promising application in diagnoseof clinical suspected cases.