| This study was divided into two parts.The first part was a comparative study of five commercially available reagents for the extraction of viral RNA and comparison of the effects of different methods on the extraction of viral nucleic acids.The second part established a fluorescence quantitative method for the detection of nucleic acids in pig saliva and its preliminary application.1?Select commercially available 3 cell lysates(RNAiso Plus,TRNzol Plus,and RNAOUT)and 2 DNA/RNA co-extract kits(RNA-DNA double extraction kit,rapid DNA/RNA co-extraction kit)to extract pigs RNA of PRRSV JXA1-R strain,swine fever virus(CSFV)HCLV strain and Japanese encephalitis virus(JEV)SA-14-14-2 strain directly quantified by RNA and Real-time PCR indirectly evaluates the quality of the extracted RNA.Direct RNA detection results showed that the RNA concentration obtained from RNAiso Plus,TRNzol Plus,and RNAOUT extracted from three cell lysates was relatively high;real-time PCR indirect detection results showed that the highest level of RNA was found in the rapid DNA/RNA co-extraction kits.,RNAiso Plus and TRNzol Plus 2 cell lysates were followed.Studies have shown that RNAiso Plus,TRNzol Plus,and RNAOUT can be used for clinical RNA virus detection and quantitative analysis.Porcine circuitous disease mainly circuitous type 2 pathogenic,causing disease in pigs.For instance,systemic wasting syndrome,reproductive failure in sows and piglets and other central nervous system disease and so on.The circuitous can destroy the immune system of the pig and cause immunosuppression.The porcine circuitous disease is mainly infected pigs and infected pigs.It is a type of viral infection characterized by transmission of the virus through the respiratory tract and digestive tract.Clinical manifestations are more complex.2?The porcine circovirus disease is mainly caused by circovirus type 2,resulting in diseases such as multiple system exhaustion syndrome after weaning of pigs,reproductive failure of sows,and central nervous system of piglets.Saliva contains pathogens and antibodies.Since the saliva detection of pigs has the characteristics of simple collection method,low cost,and non-irritating to pigs,it has attracted more and more attention and applications.The purpose of this experiment is to establish a method for saliva detection,combining fluorescence quantitative PCR with saliva detection,and establishing a fluorescent quantitative method for detecting PCV2 in saliva.The saliva collection method and the nucleic acid extraction method in saliva are improved,and the established fluorescence quantification method is only for the PCV2 type.In this experiment,a specific gene fragment was amplified,combination plasma was constructed,and combination plasma was used as a template to draw a standard curve of a fluorescence quantitative method.Establishing a fluorescence quantification method through a series of experiments such as specificity experiments,sensitivity experiments,and producible experiments shows that this method has the characteristics of strong specificity,good dependability,and high sensitivity.The established method for quantitative detection of PCV2 in saliva was 1000 times more sensitive than the conventional PCR method.The feasibility of establishing PCV2 fluorescence quantification method in saliva was further verified by clinical samples.In this experiment,33 samples of saliva were collected from 4 different pig farms in Jungian during the period from October to November 2017.Common PCR was performed on the collected samples.Fluorescent quantitative PCR assays and comparative test results showed that the positive detection rate of each pig farm was increased,and the PC2 infection status of pig farms could be more directly demonstrated.Through the application of clinical samples,the potential of using saliva as a test sample for disease antigen detection was demonstrated,and the saliva test was further provided. |
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