| In this study, reverse transcription nested polymerase chain reaction (RT-nested PCR) technique was successfully developed for detection bovine viral diarrhea-mucosal disease virus (BVD/MDV).Two pair of primer were designed using computer and synthesized respectively, according to 5 ' untranslated cDNA region of the NADL stain BVDV and classical swine fever virus (CSFV).Viral RNA was extracted, reverse transcripted and amplified by PCR and nested PCR. Using corresponding primer P, and P2 , BVDV and CSFV could be detected by RT-PCR. When analyzed by agarose gel electrophoresis, a single band with the expected size of about 280bp was abstain. When primer P3 and P4 was used, only three stains BVDV were amplified by nested PCR, a single band with size of about 191 bp was detected by agarose gel electrophoresis, CSFV wasn't amplified.51The result was expected. No amplification was observed when using RNAs extracted from Pseudorabies virus (PRV) and uninfected MDBK cell. 10 TCID50 BVDV could be detected by RT-PCR, and 10-1 TCID50 BVDV could be detected by nested PCR. The result demonstrated that the RT-nested PCR was not only a specific, sensitive, conveninent and rapid method for diagnosis of BVD, but also a way to differentiate BVDV and CSFV.In addition, other aspects of the RT-nested PCR technique for detecting BVDV, such as preparation and store condition of template RNA and the techniques for diagnosis BVD were tested. |
