Study On The Development Of A RT-PCR ELISA Method And The Diagnositic Kit On The CSF

The classical swine fever (CSF) which is prevailing in pig breeding countries and harmful to the development of hog industry, is one of the key epidemic diseases that are quarantined and prevented strictly by every country in the world. Therefore, it is necessary to develop a rapid, simple, high standardized diagnostic technique that can be applied in the clinical detection of the classical swine fever virus (CSFV) to meet the development of hog industry and market demand. In this experiment the specific primer and capture probe was designed according to the 5'-UTR of the CSFV, and the product of the reverse transcription polymerase reaction (RT-PCR) was labeled by digoxingenin (DIG).Then the RT-PCR ELISA method was established to hybridize and capture the dig-labeled product of the RT-PCR with the probe which had been binded to the microtiration plate. During the course of the RT-PCR ELISA development, some works are carried out such as follows: extraction of total RNA and amplification of C strain, Shimenqiang strain and infectious materials by RT-PCR; definition of the PCR reaction conditions; detection of the linked-ratio of microtiration plate; determination of positive or negetive judgement standard; determination of the most suitable the block concentration; the suitable concentration of the Streptavidin; determination of the suitable ratio Streptavidin and Anti-dig-POD; determination of the most suitable ratio of Streptavidin and probe; definition of the most suitable hybridation conditions; definition of standard positive control; cross experiment; sensitive experiment; specific experiment; preservation course and field experiment etc. The results are as follows: (1) the block solution is the PBS solution containning BSA and Tween-20 the percentage of which are 0.2% and 0.1% respectively. (2) the most suitable Streptavidin coated concentration is 1:960. (3) the most suitable Anti-dig-POD concentration is 1:3000; (4) the most suitable probe concentration is 1:80; (5) the most suitable hybridation condition is 37CC 2h; (6) no cross reactions among various virus; (7) 100 times more sensitive than the agarose gel detection.Through the detection of the CSFV standard strain(C strain, Shimenqiang strain etc.) and wild various infections, a new CSFV detection method was established. This method which can save time and be used conveniently in practice has strong specificity, high sensitivity, no radioactivty. So it provides a new way for researchers on the molecular epidemiologic investigation and early diagonosis of CSFV.