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