Differentiation Between Vaccine Strain and Field Isolates of Classical Swine Fever Virus Using Polymerase Chain Reaction and Restriction Test / 微生物学通报

A nest RT-PCR/restriction test has been developed in order to distinguish the lapinised vaccine strain from field isolates of classical swine fever virus. The restriction enzyme cut sites mapping of the major coding sequence of E2 gene lapinised vaccine strain and ShiMen strain of classical swine fever virus have been compared. Ten and sixteen unique restriction markers have been found in the lapinised vaccine strain and ShiMen strain. The restriction enzyme cut sites mapping of the twenty six unique restriction marker in the major coding sequence of E2 gene of 17 classical swine fever field isolates have been analyzed. Only 3 sites (HgaI、Hin8I及Hsp92I) are present in the lapinised vaccine strain sequence. Two pans of nested primers and a criteria of analysis have been designed for HgaI restriction marker site. The tests have been conducted first on the lapinised vaccine strain and ShiMen strain of classical swine fever virus resulting in predicted restrection patterns. Finally, the tests have been applied to 5 field isolates of different gene group analyzed by phylogenetic study. The result showed that only HCLV strain gene can be cut to 2 fragment by Hgal , and ShiMen strain and 5 field isolates cant be cut At the same time the sensitivity and specificity of nest RT-PCR have been tested. The sensitivity is 0. 2MLD. The specific fragment of BDV and BVDV were not obtained by the nest RT-PCR. These results showed that the development of the nest RT-PCR/restriction tests is very important for the control and perish of classical swine fever in china.

RESEARCH ON THE TESTING OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS BY NESTRT-PCR / 微生物学通报

Three primer were designed based on the consevered area of the genetic of the ATCC VR-2332 strain and LV strain. And the nest RT-PCR of testing porcine reproductive and respiratory syndrome virus were developed. The nest RT-PCR against ATCC VR-2332 strain, LV strain and B13 strain were done by this method.The DNA fragment were obtained specially from the three strains isolated from different region. The size were 430bp (430bp) , 410bp (413bp) and 410 bp (413bp) separately. But the DNA fragment were not obtained from HCV, PPV and PRV. Its sensitivity was 10-2 TCID50. It's sensitivity increased 10000 times than one step RT-PCR. It should make the method of testing porcine reproductive and respiratory syndrome virus more sensitive, fast and accurate.