Estblish Multiplex PCR Diagnosis Method And A Combined Detection Mieroarray Diagnostic Method For Five Reproductive Virus Disease Of Swine

The increase in the density of large-scale swine herds has increased the frequency of contact between herds and indirectly accelerated the spread of disease.China's swine blight is also increasingly complicated and serious,and the proportion of mixed infections has increased significantly.Among them,classic swine fever virus(CSFV),porcine parvovirus(PPV),Porcine reproductive and respiratory syndrome virus(PRRSV),Japanese encephalitis virus(Japanese encephalitis virus).,JEV)and porcine circovirus II(PCV-2)caused porcine reproductive disorder diseases as the main hazards.Because five kinds of reproductive disorder diseases have similar clinical symptoms and are often mixed infections,the difficulty in clinical rapid diagnosis is increased.At present,the common methods of serological diagnosis and virus isolation and identification in laboratories are time-consuming?low-sensitivity,and cannot meet the needs of clinical diagnosis.Gene chip technology is a new gene detection method integrating molecular biology technology,computer technology,and fluorescent labeling technology.This method is characterized by rapidity,high specificity,high sensitivity,high throughput,and diversity,that has been widely used in disease diagnosis,drug screening,polymorphism analysis and other fields.The multiplex PCR method has the characteristics of being able to detect multiple pathogens at one time.At the same time,this method has high specificity and high sensitivity,which is more suitable for the rapid diagnosis of mixed infections.In this study,based on the incidence of swine diseases in China,specific primers and probes were designed for the conserved regions of the above five viruses,a multiplex PCR diagnostic method capable of simultaneously detecting five viruses and a combined detection microarray diagnosis method were established.The tow method was initially applied to clinical testing.The main findings are as follows:1.Establishment of multiplex PCR diagnostic methods for CSFV,PPV,PRRSV,JEV,and PCV2: According to the Gen Bank CSFV,PPV,PRRSV,JEV,and PCV2 genome sequences,Primer Premier 5.0 software was used to design 5 pairs of specific primers for amplifing the CSFV E2 gene,PPV VP2 gene,PRRSV N gene,JEV C gene and PCV2 Cap gene.Establish a multiplex PCR reaction system on the basis of a better single PCR amplification.At the same time verify the specificity,sensitivity and stability of this method.The specific test results showed that the establishment of this method can amplify specific fragments of the above five viruses at one time,and no amplification of other pathogens;sensitivity test shows that the sensitivity of the method reaches 10-3ng level and reproducible.Using this method to detect 16 clinical samples at the same time,the results showed that 14 of the 16 samples were positive and the mixed infection rate was 21.4%(3/14).The mixed infection of PCV2 and PRRSV was more serious,reaching 14.3%(2/14).2.Construction of a combined detection microarray diagnostic method: specific primers and probes were designed based on the conserved regions of the above five viruses,and the probes were cured by spotting on an aldehyde-based substrate according to a predetermined procedure and post-processed to prepare the oligosaccharides nucleotide chip.The amplified multiplex PCR reaction system was used to amplify the nucleic acid fragment that was complementary to the probe and labeled with Cy3 fluorescence.After hybridization,the fluorescent signal on the chip was scanned and analyzed to determine the result.At the same time,it was established that the best hybridization reaction conditions were 55°chybrid box hybridization for 2h,and a good hybridization signal was obtained when the probe concentration was 1?M.Sensitivity test results show that the diagnostic method has reached 10-5ng level,and the coincidence rate between clinical test results and multiplex PCR results is 100%.