| With the intensived and industrialized development of pig industry,epidemic prevention and control has always been one of the important topics for the healthy development of pig industry.In the prevention and control of epidemic diseases,the timely diagnosis and prevention of respiratory diseases is the top priority.Although immunization has been popularized,the epidemic still threatens the pig industry.And the disease outbreaks are mostly characterized by the simultaneous or successive occurrence of several diseases.Therefore,timely and rapid diagnosis is the most effective prevention and control measure for the discovery and control of diseases.Some respiratory diseases that may lead to a large-scale epidemic in pigs have become the focus of prevention and control.Porcine pseudorabies virus,porcine circovirus and porcine reproductive and respiratory disorder virus often show mixed infection in the clinical breeding of pigs,and the infection of PCV2 and PRRSV will lead to immunosuppression in pigs.In addition,PRRSV and PRV show similar symptoms.Therefore,how to accurately diagnose these pathogens quickly and accurately,so as to quickly prevent and control the epidemic disease,is of great significance to the development of China’s pig industry.TaqMan probe fluorescence quantitative PCR detection method has the advantages of high sensitivity and high specificity compared with other traditional detection methods due to the addition of probes.It can be widely used in clinical pathogen diagnosis.This study intends to establish a multiple real-time quantitative PCR detection method of PRV,PCV2 and PRRSV,so as to realize the rapid diagnosis of mixed infection of PRV,PCV2 and PRRSV in clinical.For this reason,we conducted the following research:(1)A single fluorescence quantitative PCR method for the detection of PRV,PCV2 and PRRSV was established.Specific primers were designed according to the relatively conservative gene sequence regions of UL27 gene of PRV,cap gene of PCV2 and ORF7 Gene of PRRSV,and the fragments of the amplified target gene were cloned into p UC19 empty vector,so as to construct the recombinant plasmid standard of each virus.Then,the gradient dilution standard was used for single fluorescence quantitative PCR amplification,and the standard curve was made according to the amplification cycle.Finally,the single fluorescence quantitative detection method of each virus was constructed.In addition,the sensitivity of PRV,PCV2 and PRRSV was 32 copies /μL、23copies/μL and 51 copies/μL.In addition to high sensitivity,the method also has strong repeatability and specificity.(2)A multiplex fluorescence quantitative PCR method for the detection of PRV,PCV2 and PRRSV was established.According to the reaction system and reaction conditions of the single fluorescence quantitative PCR method constructed by each pathogen,using the above constructed standard as the template,after adjusting the reaction conditions and system,the multiple fluorescence quantitative PCR detection methods of PRV,PCV2 and PRRSV were established.The results showed that the lower detection limits of PRV,PCV2 and PRRSV were 71,35 and 67copies/μL respectively.The coefficient of variation within and between groups of parallel detection was not greater than 1%.It is proved that this method not only has good sensitivity,but also has strong reliability.(3)The established multiple TaqMan fluorescence quantitative PCR method was used to evaluate the application effect of clinical detection.It was found that the positive detection coincidence rate of PRV,PCV2 and PRRSV pathogens was more than 91.94% compared with the national standard conventional PCR gel electrophoresis method widely used in clinical.It is proved that this method has a good clinical application prospect.In short,the single and multiple fluorescence quantitative PCR methods of PRV,PCV2 and PRRSV established in this study can quickly and accurately detect these common diseases in pigs,and have high clinical application value. |
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