Establishment of Multiple RT-PCR Diagnostic Techniques for Avian influenza virus (AIV), Newcastle disease virus (NDV), Classical swine fever virus (CSFV) and Food and mouth disease virus (FMDV)

The homology of the sequences, reported and registered in GenBank of different strains of Avian influenza virus(AIV), Newcastle disease virus(NDV), Classical swine fever virus(CSFV)and Food and mouth disease virus(FMDV), was respectively analyzed and compared with each other. According to the properties of these viruses, the conservative domain of M gene for AIV, F gene for NDV, 5′non-coding domain end for CSFV and 2B gene for FMDV were selected for PCR amplification. In order to prevent the formation of conformational dimers between different primers, four pairs of primers designed with the DNAsis system under the condition of G+C(50%~60%), 18~25 bp in length and Tm (72~85), were analyzed with VNTI5.5 system. The specific fragments amplified were as follows: 141 bp for FMDV, 200 bp for CSFV, 319 bp for NDV and 471 bp for AIV. The optimal conditions of PCR for each virus mentioned above were determined by orthogonal assay, and 2 or 4 of the 4 pairs of primers were then combined and used for amplification trials. The results showed that four specific fragments in different length would be successfully amplified in one tube at the same time. The products of PCR were tested to be specific by sequencing. Of 46 pathological samples detected with the multiple PCR, five were AIV, seven were NDV, fifteen were CSFV and six were FMDV. The amplifications above were identified by single PCR. Meanwhile, the results were corresponded to those of electronic microscopy, HA and ELISA assay. The method described here was very practicable and sensitive, specific, simple and cheap. It could be used in diagnosis of AIV, NDV, CSFV and FMDV for different animals.