The Establishment Of Rapid Detection Method And Epidemiological Investigation Of Porcine Cytomegalovirus In Regions Of Henan Province

The Porcine cytomegalovirus disease caused by Porcine cytomegalovirus (PCMV) is a conditional infectious disease. The research of this disease is not deep for a long time, so the danger to the herd does not get enough attention. But recent studies have found that the virus mainly proliferate in porcine alveolar macrophage, destruct the body’s immune system, resulting in reduced animal’s resistance after infection, which caused to complicated and secondary infection with other pathogens (such as PRRSV), the harm of Porcine cytomegalovirus to the pig industry gradually attracted attention of industry professional. Therefore, the study on rapid diagnostic method and the epidemiological investigation of the disease will have great significance to reduce the harm of the pig industry.In this study, DNA polymerase gene of PCMV was cloned and determined by genetic engineering technology, and compared a lot of PCMV DNA polymerase gene sequences in GenBank, then specific primers and TaqMan fluorescent probes to establish PCR and real-time PCR (FQ-PCR) were designed and synthesized, TaqMan fluorescent quantitative real-time PCR (FQ-PCR) to detect PCMV after optimizing the reaction conditions was established, and the sensitivity, specificity and reproducibility of PCR and FQ-PCR were carried out; The suspected samples in clinical were detected by using FQ-PCR method compared with the ordinary PCR and the viral content of the different tissues organs and serum in the porcine cytomegalovirus-positive pigs were quantified; An epidemiological survey of the porcine cytomegalovirus infections and mixed infection in regions of Henan Province was conducted from2009to2011.The results indicated that the DNA polymerase gene of PCMV was successfully cloned and determined, compared with landing other PCMV polumerase gene, the nucleotide homology was99.3%. Besides PCR and FQ-PCR methods for testing PCMV were successfully established, which had high specificity, high sensitivity and good repeatability. The minimum detection limit of100copies/μL. There was a good linear relationship between the cycle threshold of the standard curve and template concentration in the established FQ-PCR method, and the correlation coefficient was0.998; the detection sensitivity of FQ-PCR method was up to1copy/μL, which was100times than conventional PCR; The specificity of the method was high, and it rendered positive response curve on amplification of pGEM-T/PCMV recombinant plasmid, while negative reaction curves on the amplification of6control pathogens; With6times repeated amplification of different concentrations pGEM-T/PCMV recombinant plasmids, respectively, the results were well. We applied the method to detect15copies of histopathological materials with clinically suspected PCMV infection, in which,9samples were positive, the positive coincidence rate was100%for detection with conventional PCR method. Different internal tissues organs and serum for PCMV infection positive pigs have virus. That is the highest PCMV amount of the internal tissues organs in pigs was tonsils, and the lowest was small intestine.An epidemiological survey of PCMV infection status in swinery was conducted from2009to2011. The results indicated that the infection of porcine cytomegalovirus was increasing year by year, and the positive rate was highest in2011(58.7%), followed in2010(52.6%), and the lowest rate was in2009(44.3%). At the same time, the results of epidemiological investigation on PCMV infection were various in the different scale farms.Firstly,the positive rate was the most highest in the pigs of scatter-feed (60.5%), secondly, in small-scale field(50.5%), thirdly, in large-scale field(33.3%). The investigation of epidemiological investigation on PCMV infection in different ages showed that porcine cytomegalovirus infection was common in all ages of pigs. Piglets(65.2%) was one of the highest positive rate, then sows(47.9%), and then fattening pigs(37.9%). According to the epidemiological investigation, there were the evidence of duplicate infection(PCMV+PRRSV) and ternary infection(PCMV+PRV+PCⅦ)In this study, the PCR and FQ-PCR detection method of PCMV was successfully established, The content of PCMV in different tissues organs and serum was quantified, an epidemiological survey of PCMV infection in regions of Henan Province, and mixed infection with other pathogens among swinery was conducted by using the FQ-PCR method.The study enriched the content of PCMV epidemiological investigation, depened the understanding of the status of China’s PCMV infection, and will have important guiding significance in scientific control and prevention of the PCMV.