| As an important part of the modern food structure and food industry,the quality,safety and hygiene of meat has attracted widespread attention.As the main meat produced and consumed in my country,pork is particularly safe.Porcine epidemic diarrhea virus(PEDV)is the main pathogen causing the outbreak of porcine viral diarrhea,which has brought great losses to my country’s pig industry.Only by establishing accurate and efficient detection methods and taking corresponding measures in a timely manner can the safety of pork food be effectively guaranteed from the source.Loop-mediated isothermal amplification(LAMP)and Quantitative PCR(qPCR)are commonly used for molecular biological detection.However,these methods are prone to pollution,time-consuming and need low temperature transportation and storage.In this study,by establishing a LAMP detection method for PEDV,screening and optimizing lyophilized excipients,exploring and optimizing the lyophilization process,and initially establishing a PEDV LAMP lyophilization detection system,PEDV can be quickly and efficiently diagnosed on-site.The research results are as follows:(1)Establishment of a real-time fluorescence quantitative LAMP(qLAMP)method for porcine epidemic diarrhea virus.Specific primers were designed and screened for the N protein gp6 gene of PEDV;Mg2+ concentration,bataine oncentration,temperature and other factors were optimized to establish a qLAMP method;the sensitivity,detection limit,specificity and repeatability of the system were verified and the actual sample for testing.The results showed that the sensitivity was 1 fg/μL,and 157 copies/μL of plasmid DNA could be detecte;there is no amplification reaction with pathogens such as respiratory syndrome virus;the coefficient of variation of the repeatable experiment was less than 3%;the positive detection rate of samples was close to that of qPCR,and the detection of the sample can be completed in a short time.(2)Screening and optimization of lyophilized excipients.Lyophilized protective agents and excipients were screened,through single factor and response surface experiments,the optimal formulation of lyophilized excipients was obtained.The results showed that the addition of excipients was 9.0%raffinose,3.0%mannitol and 1.5%bovine serum albumin,and the actual operation was carried out according to the optimal process conditions,and it was confirmed that the theoretical predicted value was close to the measured value;the order of influence was:raffinose>mannitol>bovine serum albumin;the lyophilized PEDV LAMP detection reagent has a smooth and plump appearance and can be dissolved quickly;the selected formula can effectively reduce the damage to the active components of the system by freeze-drying.(3)Establishment of a freeze-dried LAMP detection system for porcine epidemic diarrhea virus.The LAMP detection system(except betaine)with freeze-dried auxiliary materials were pre-mixed,key process parameters were explored,freeze-drying procedures were optimized,and finished product performance and storage stability were evaluated.The results showed that the eutectic temperature of the system was-39.8℃,and the freeze-drying cycle was 32 h;the detection limit of the plasmid was 157 copies/μL,with good specificity and stability;it was stored at 37℃ for 2-3 days and stored at 25℃ 30 days;the storage time model of the detection system was established by first-order chemical reaction kinetic equation and Arrhenius equation,and the storage time of the system at 4℃ was predicted to be 369 days.To sum up,in this study,a solid LAMP detection system was prepared by using freeze-drying technology.Compared with the liquid detection system before freeze-drying,the on-site operation steps were simplified,and the requirements for storage and transportation of LAMP detection reagents were effectively reduced,which is suitable for on-site rapid detection and provides a technical basis for the further promotion of detection reagents. |
PDF Full Text Request