Preliminary Study On Application Of The Drug Certificates Of Fluorescent Quantitation PCR Detection Kit Of Porcine Circovirus Type 2

According to market research,pork is the most demanded meat food.Porcine Circovirus Type 2(PCV2)in Porcine Circovirus Type(PCV)is one of the disturbing factors affecting the growth,development and reproduction of pigs.Porcine circovirus type 2 is the main pathogen causing Post-weaning Multisystemic Wasting Syndrome(PMWS)in pigs,thus bringing huge economic losses to the pig-breeding industry.Therefore,it is necessary to develop a qualitative and quantitative method for rapid detection of PCV2 in pigs.The laboratory has established the rapid qualitative and quantitative detection technology of PCV2 fluorescent quantitation PCR,and developed the kit with the technology.It is named circovirus type 2(PCV2)fluorescent quantitation PCR rapid detection kit,which is currently apply for drug certification.In this experiment,fluorescent quantitation PCR technology was used to qualitatively and quantitatively detect PCV2,and the virus content was quantitatively detected by making a standard curve.This method can be used to determine the contents of PCV2 in infected pigs,and to detect,isolate and eliminate piglets.This thesis mainly focuses on the raw materials,production process and reaction system of the kit.The main contents and results are as follows:In the study of raw materials of detection kit,Taq enzyme,PCR reaction buffer and UDG enzyme produced by three companies were compared and optimized.Among the results of qPCR amplification,the Taq enzyme and PCR buffer produced by Takara bio-engineering(Shanghai)co.,Ltd are the highest fluorescence growth value and Ct value.The UDG enzyme produced by Shanghai Biocolor BioScience and Technology Co Ltd is of high quality and low price.At the same time,the references are prepared for the kit.The Ct value of the positive reference is 20-25,the Ct value of the lowest detection limit reference is 30-35,the Ct value of the precision reference was 20-25,and the negative reference had no amplification curve and Ct value.In the research on the production process of the kit,the factors affecting the production process temperature were studied,that is,the optimal storage period of samples with high concentration,medium concentration and low concentration were studied in freeze-thaw times,2-8?,-20?,-70? and low-temperature transportation,respectively.The nucleic acid concentration of the medium concentration' samples under tests would be decrease after repeated freezing-thawing at-20?,and the growth value of fluorescence would decreased from 4.13 to less than 1 after the numbers of freezing-thawing exceeds 5.Therefore,it is generally recommended that the numbers of freezing-thawing should not exceed 5.In same principle,different concentration of samples simulated low temperature transport of ice packs at room temperature thought 0 hours(hour,h),24 h,48,72 h,96 h and 120 h,its nucleic acid concentration and Ct value of the samples transported 72 h,the CV value is less than 5%,and the low concentration samples the Ct value in the transport 96 h is slower,Coefficient of variation is more than 5%.Therefore,comprehensive consideration should be given to the low temperature transport of samples in ice packs should not exceed 96 h.Different concentrations of samples in the 2-8? though 0 day,1 day,2 days,3 days,4 days and 5 days extraction and qPCR nucleic acid amplification tests respectively,high concentration samples were less than 5% but when the medium concentration samples were stored at day 5,its coefficient of variation was greater than 5%,variation coefficient of Ct value at day 4 of low-concentration samples was more than 5%,so 3 days was used as the validity period of samples stored at 2-8?.Nucleic acid extraction and qPCR amplification were performed when samples of different concentrations were stored at-20? for 0 day,1 month,2 months,4 months,6 months,8 months,10 months and 12 months,respectively.Similarly,6 months was used as the validity period of samples stored at-20?.Nucleic acid extraction and qPCR amplification were performed when samples of different concentrations were stored at-70? for 0 day,1 month,2 months,4 months,6 months,8 months,10 months and 12 months,respectively.Similarly,12 months was used as the validity period of samples stored at-20?.In the related dosage test of the total reaction system of the kit of 12.5?L,25?L and 50?L,respectively.The results of the 25?L and 50?L total reaction systems showed that the samples all obtained high fluorescence signal values.In the 12.5?L total reaction systems,the sensitivity was not high and the fluorescence growth value was lower.The high sensitivity of the 50?L reaction system would easily lead to false positives and cost twice as much reagent.Therefore we're going to regard 25?L as the total reaction system.