Establishment And Application Of A MPCR Method For Simultaneous Detection Of Porcine Respiratory And Viral Diseases

With the development of large-scale and intensive pig industry, pathogen mixed infection and secondary infection of pig diseases are becoming more and more common.Respiratory disease as one of the major diseases is often occurred in recent years in scale pig farms of China, causing great losses to our country. Pseudorabies virus, porcine cricovirus, reproductive and respiratory syndrome virus, swine fever virus, porcine parvovirus, and swine flu virus are the most common viral pathogens of pig respiratory diseases, and often show mixed or secondary infection in clinical. Infection caused by multiple pathogens in pigs is becoming more and more poplar nowadays, and that caused by two or more pathogens is leading to high morbidity and mortality and making more difficult in the diagnosis and treatment of diseases. m PCR differential diagnosis methods for these pathogens are established to rapid detect mixed infection in clinical, and provides an effective technical means for rapid diagnosis and prevention and control for related diseases. The main research contents are as follows:1、Establishment of the Single PCR/RT-PCR method of PRV, PPV, PCV2, PRRSV, CSFV and SIVAccording to the gene sequences published in Genbank of PRV g B（AF257079）, PCV2 ORF2（NC₀05148）, PRRSV ORF6（NC₀01961）, CSFV C（AY805221）, PPV NS1（NC₀01718）and SIV M（GU086140）, six pairs primers were designed and synthesized to amplify part fragments of the specific genes of PRV g B, PCV2 ORF2, PRRSV ORF6, CSFVC, PPV NS1 and SIV M respectively and the corresponding amplified fragments are 192 bp, 255 bp, 364 bp, 530 bp, 759 bp and 981 bp,successively. Then single PCR/RT-PCR method of PRV, PPV, PCV2, PRRSV,CSFV, PPV and SIV were established by optimizing the reaction conditions to lay foundation for the establishment of multiplex PCR.2、Establishment and Preliminary Application of the 3-plex PCR and 3-plex RT-PCR methodOn the basis of the established single PCR method of PRV, PCV2, PRRSV, CSFV, PPV and SIV, the 3-plex PCR and 3-plex RT-PCR reaction condition was optimized and the multiplex PCR and RT-PCR method wree established. The results showed great sensitivity and specificity and the detection result were both best at 56℃. The preliminary application test showed the detection results of clinical samples were consistent with that of single PCR and RT-PCR.3、Establishment and Preliminary Application of the 4-plex PCR method, 5-plex PCR method and 6-plex PCR methodOn the basis of the established 3-plex PCR and 3-plex RT-PCR method, the 4-plex PCR method, 5-plex PCR method and 6-plex PCR method reaction condition was optimized and the three multiple PCR methods were established. The results showed the multiple PCR assays can detect only the aim virus, and the detection viral nucleic acid level is pg. The results of preliminary application for 48 clinical samples showed the established multiple PCR assays has wide prospects.