Development And Application Of Visual Enzymatic Oligonucleotide Microarray For Simultaneouly Detecting PEDV,TGEV,GAR And PDCoV

Porcine epidemic diarrhea virus(PEDV),Transmissible gastroenteritis virus(T GEV),Group A rotavirus(GAR)and Porcine Deltacoronavirus(PDCoV)are the main pathogens causing viral diarrhea in piglets.These four types of piglets have very similar clinical manifestations of diarrhea and are difficult to differentially dia gnose.In this study,PEDV,TGEV,GAR,and PDCoV four kinds of diarrhea viru ses were combined,and a set of PEDV-TGEV-GAR-PDCoV enzymatic enzymes w as constructed by combining the enzymatic reaction color-developing technique,asy mmetric PCR single-strand amplification technology,and gene microarray technolog y.Co-examination visualization of gene microarray technology,preliminary clinical evaluation evaluation.The results showed that the optimal concentration ratios of upstream and dow nstream primers for asymmetric PCR of each target gene were:PEDV-S:1:20;PE DV-M:1:10;TGEV-S:1:20;TGEV-N:1:20;GAR-VP7:1:10;GAR-NSP4:1:10;PDCoV-M:1:10;PDCoV-N:1:20,the target fragment has the most single-stranded product.The operating procedure for microarray detection was determined as foll ows:after 90 min hybridization at 50°C,washing was performed.The result was read with a 2,000-fold diluted streptavidin-labeled horseradish peroxidase enzyme and visualized with a DAB coloring solution.In this study,tan spots were generat ed according to the positive control,and no spots were generated in the blank co ntrol.The quality of the microarray detection and the criteria for determining the results were determined.The specificity of this visualization co-detection microarra y was good,and the hybridization and hybridization detection results of PEDV,T GEV,GAR,and PDCoV were all positive.The hybridization results of CSFV,PR RSV,PCV2,JEV and FMDV were negative.There was no mutual interference bet ween the probes;The sensitivity of the needle gene can reach 10pg/?L(2.83×10~6copies/?l);the microarray can still be effectively detected after 180 days of stora ge.The research and application of the constructed chip to 200 samples from Sich uan and Chongqing were tested.This study successfully constructed a set of visual ization gene microarrays for high-throughput detection of four diarrheal diseases in piglets.This method is highly specific,sensitive,and inexpensive.It can meet th e needs of clinical testing and provides a clinical detection of diarrheal diseases in piglets.