Establishment And Purification Measures Of PRRSV Fluorescence Quantitative PCR

Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)can cause Porcine Reproductive and Respiratory Syndrome(PRRS).It is a highly contagious disease found in the late 1980 s,which can cause Reproductive failure in sows and severe Respiratory problems in piglets.The PRRS first erupted in the southern states of the United States in 1987,and became popular in most countries around the world.PRRS was first reported in China in 1996 and then widely distributed in our country.Highly pathogenic Blue-eared disease first outbroke in China in 2006,which was characterized by high fever,high morbidity and mortality,and which caused much loss of China's pig industry.So far,PRRSV in China's farms is mainly PRRSV,highly pathogenic PRRSV and both vaccine strains.The existence of these strains has brought great difficulty to the prevention,control and purification of PRRSV in domestic farms.Nsp4 is composed of 204 amino acids,and the molecular weight is about 21 kDa,which is a very important protease with double enzyme activity.Nsp4 is a type of virus(3C-like proteinase,3CLpro)that is encoded by all arteritis viruses and coronaviruses.To help one single pig farm to test and purify the PRRSV,this study analysed the pig disease material,and used contrast Nsp4 primers to amplify,cloned the amplification of Nsp4 gene segment in pMD-18 t carrier,and used DNAStar software to analyze the sequencing result in reference to the Nsp4 gene sequence of the PRRSV which had been found and isolated in China to compare.It is found that the similar degree of the pandemic strains in pig farms and domestic HuN4 strain gene sequences was much high after nucleic acid sequence analysis.Through the analysis of the evolutionary tree,the strains of the pig farm were similar to the HuN4 strains.Comparison of the amino acids sequences between Nsp4 sequences of the HuN4 strains and the sequencing sequences,there were individual site mutations on the sequencing sequences.To test the PRRSV of the pig farms rapidly and conveniently,this study compared pig epidemic strains with the nsp4 of HuN4 strains,and designed specific primers to PRRSV nsp4 nucleotide sequence.SYBR Green I established a fluorogenic quantitative PCR which can quickly detect the fluorescence quantitative PRRSV.SYBR Green I is a kind of dye which combines all dsDNA double helix groove area with green excitation wavelength.Under free state,the SYBR Green I showed weak fluorescence.After combined with double-stranded DNA,SYBR Green I would greatly enhance the fluorescence signal.The method could detect 10copies/?L templates at least when detecting 1010~104 copies/?L,and its specificity was strong.According to the fluorescence quantitative PCR reaction system,we amplified swine fever(CSFV),porcine circovirus(PCV),pig pseudo rabies virus(PRV),and porcine epidemic diarrhea virus(PEDV),and all the results were negative.The results was stable when tests were repeated,which showed that the method was stable,specific and sensitive.The positive rate was 100%,when SYBR Green I tested the samples of pig farms.The accuracy achieved the expected results.The test requires fewer samples and meet the diagnostic needs of PRRSV for pig farms.This paper based on the established methods of detection,summarizes and analyzes the domestic and foreign animal disease purification measures,and proposes the maximum PRRSV purification strategy according to the condition of the pig farms.The suggestions are given through the distribution of pig farms,management of pig farms,cleaning of environment,selection of introduction and immunization of vaccines so that the aim of purification of PRRSV in pig farms can be realized.