猪流行性腹泻病毒冻干qLAMP检测体系的建立

Establishment of A Freeze-dried qLAMP Detection System for Porcine Epidemic Diarrhea Virus

为建立猪流行性腹泻病毒(Porcine Epidemic Diarrhea Virus,PEDV)实时荧光定量LAMP(Quantitative LAMP,qLAMP)与冻干技术结合的快速检测方法。该实验根据PEDV的高度保守序列设计了引物并进行筛选,建立PEDV的qLAMP检测方法。筛选棉子糖、甘露醇和牛血清蛋白作为冻干辅料并优化,将最佳的配方与等温扩增试剂(除甜菜碱)混合后进行冷冻干燥处理。优化主干燥升温方式和二次干燥时间,制作出一条适合等温扩增试剂的冻干曲线,并以此制备PEDV qLAMP冻干检测试剂盒。结果显示:通过对冻干成品性状、溶解性及剩余水分含量的测定,确定了主干燥后期采用一步升温的方式进行升温,解析干燥时间为3.5 h。PEDV qLAMP冻干检测体系的最低检测限为每微升157 copies,与工艺优化前相比,其检测速率得到提升。对猪瘟病毒、猪伪狂犬病毒等5种病原无扩增反应,重复性实验变异系数均小于5%,常温条件下保存30 d,在4℃条件下至少保存12个月。该研究通过使用冻干技术建立了LAMP检测方法,具有现场操作简便快捷、检测效果好、储存时间长,适用于基层现场快速检测。

To establish a rapid detection method for Porcine Epidemic Diarrhea Virus(PEDV)using real-time quantitative LAMP(qLAMP)combined with freeze-drying technique,primers were designed and screened according to the highly conserved sequence of PEDV,then a qLAMP detection method for PEDV was established in this work.Raffinose,mannitol and bovine serum albumin were used as the lyophilized excipients and subjected to optimization,then the optimal formulation was mixed with isothermal amplification reagents(except betaine)for freeze-drying.The main dry heating method and the secondary drying time were optimized,and a freeze-drying curve suitable for isothermal amplification reagents was established,then the lyophilized PEDV qLAMP kit was prepared.The results showed that:through the determination of the properties,solubility and residual moisture content of the freeze-dried product,an one-step heating approach was adopted in the later stage of the main drying process,with the desorption drying time being 3.5 h.The lower detection limit of the PEDV qLAMP freeze-drying detection system was 157 copies/μL,and the detection rate was improved compared with that before the process optimization.There was no amplification reaction to 5 pathogens including swine fever virus and porcine pseudorabies virus,with the coefficient of variation of repeatability experiments smaller than 5%.The kit can be stored at room temperature for about 30 days and at 4℃for at least 12 months.In this study,the qLAMP detection method was established through using freeze-drying technique.The method is simple,fast and effectivefor on-site operation and the kit has a long storage time,thus is suitable for rapid on-site detection in actual operation sites.