Establishment Of Droplet Digital LAMP Pointof-care Testing Method And Its Application In Pig Enterodiarrhea Virus Detection

Porcine viral diarrhea is one of the most important infectious diseases affecting the pig breeding industry,among which Porcine viral diarrhea is the most serious,with rapid spread,high incidence and severe symptoms,causing huge economic losses to pig breeding enterprises.To solve the above problems,this study was conducted based on the results of a study conducted in the United States.To solve the above problems,this study established a droplet digital LAMP Point-of-care detection methods and platform based on microdroplet digital nucleic acid detection technology using Loop-mediated isothermal amplification（LAMP）,and the main research contents and results are as follows:1 Construction of an acoustic wave-driven droplet generation platformAcoustic waves can drive the vibration of a glass capillary with a stretched tip to cause the water phase inside the capillary to spontaneously inject from the tip into the oil phase to form a water-in-oil monodisperse droplet.Based on this,a tip-stretching glass capillary with a piezoelectric sensor was used to make a vibrating tip device,a droplet collection cell was prepared using polydimethylsiloxane（PDMS）and hydrophobic slides,and commercially available components such as portable microscopes and multi-axis combination tables were designed and purchased to assemble and construct an acoustic wave-driven droplet generation platform,optimize the oil phase composition,and characterize the droplet generation performance of the platform.The main results are as follows:（1）The constructed acoustic wave-driven droplet generation platform is disassembled,occupies little space,and is simple and low-cost to operate.（2）Suitable oil phase（mobile phase）composition:mineral oil with 5%Gransurf 90surfactant.（3）The platform can generate stable and homogeneous monodisperse droplets（coefficient of variation CV<3.3%）in a wide size range,flexibly,accurately and on-demand in real time,with the correlation coefficient R²=0.9984 between droplet volume and amplitude frequency.2 Establishment of the droplet digital LAMP Point-of-care detection methodsThe droplet digital LAMP Point-of-care detection methods was developed and validated based on an acoustic wave-driven droplet generation platform.Using the p MD18-T plasmid vector,the N-gene recombinant plasmid of porcine epidemic diarrhea virus（PEDV）strain CV777(concentration 1.74×10¹⁰ copies/μL)was constructed by TA-cloning,and the appropriate LAMP primer set was designed and screened based on the conserved sequence of N gene,and the reaction temperature was optimized to test the specificity,sensitivity and reproducibility of the metgenehod and to applied to the detection of porcine epidemic diarrhea（PED）positive material,and the main results were as follows:（1）The droplet digital LAMP instant assay could achieve the detection of PEDV at65°C and 1 h,and the results were consistent with those of PCR.（2）The four porcine diarrhea-causing viruses PEDV,TGEV,PRRS,PDCo V and the negative control were detected by this method,and only the PEDV group was amplified with good specificity.（3）The method successfully detected four concentration gradients of PEDV N plasmid DNA with a correlation coefficient R²=0.9998,and the lowest concentration detected was41.6 copies/μL.（4）The method was repeated between groups（3 concentration gradients,3 sets of replicates for each concentration gradient）and within groups（same concentration template,different times of detection）,and the CV of both replicates was<5%,with good reproducibility.（5）The result of using this method for PED-positive stool samples was 5447.4copies/μL.3 Construction of the smartphone optical droplet detection platformWe used Solid Works software to design the support platform and 3D print it,purchased optical components such as lenses and filters,assembled and constructed the optical droplet detection platform for smartphones,tested and optimized the imaging performance of the platform,applied it to the droplet image acquisition for the detection of PED-positive stool disease results by the droplet digital LAMP method,and wrote the program for processing the droplet images,and the main results are as follows:（1）The constructed smartphone droplet optical detection platform is portable and low-cost.（2）The platform can distinguish between positive and negative droplets,and the droplet images acquired using a smartphone are clear.（3）The macro code written based on Image J software can automatically and batch process the droplet bright-field and fluorescence images,and the error between the results and the manual processing is only 0.4%.In summary,we constructed an acoustic wave-driven droplet generation platform and developed a droplet digital LAMP Point-of-care detection methods based on this platform,and then designed a smartphone optical droplet detection platform for image acquisition of detection results,and wrote macro codes based on Image J software to automatically and batch process the data and analyze them.