Optimization And Application Of Microarray Detection Method Shared Detect The Three Diarrhea Viruses

As the rapid development of pig raising industry in China, large scale farming has become inevitable trend, which lead a complicated manifestation of swine diseases and serious phenomenon of mixed and secondary infections. Porcine Rotavirus (PoRV), Porcine Epizootic Diarrhea Virus(PEDV) and Porcine Transmissible Gastroenteritis Virus(TGEV) are three normal viral diseases that cause piglets serious diarrhea in scaled pig-raising industries in China. Above all, it is of great importance to construct a gene chip detection method to detect the three diseases at the same time.1. preparation and quality control of shared detection microarrayIn this research, conditions of point system were optimized and identified by hybridizing and scaning.The selection of sample liquid and best concentration of sampling were determined, and by comparing the two sample liquids of CapitalBio and BaiO company as well as the best mixture ratio, it was shown that it was eligible when mixed with 1:1. The best sampling concentration was determined to be 600 ng/μL by hybridizing with probe PM diluted to a certain concentration.The best dealt condition was also determined in this research, with contact printing, the probe was spotted to amino-modified glass surface, and by comparing different hydration time, selection of wash liquid and drying method, the final pointing system was determined as fellow:4s hydration in 60-80℃ water,30min UV crosslinking,8h drying at 80℃, and centrifuging immediately after washing with 0.2% SDS, and stored in airtight container.Through quality control of microarray preparation, lattice points of point system held advantages of equal size, low initial background value and high signal value after hybridization, all of which laid a basis for optimization and application of further hybridizing conditions.2. optimization of shared detection microarray assay of swine diarrhea virusesIn this research, condition of target genes’hybridization was optimized and also identified by hybridization and scan.The selection of target gene marking was determined, and by comparing directly marked primers and indirectly marked among C bases, it was shown that target genes with modified Cy3 in 5’was better in hydration effect and signal value than target gene marked with Cy3-dCTP after hydration.In this research, the selection of target gene amplification was determined by comparing with unsymmetrical PCR and general PCR, it was determined that unsymmetrical PCR with 1:40 proportion could perfectly amplified the target genes.And the best hydration temperature and time were also optimizing by hydrating with gradient time and temperature, it was found that the best condition was hydrating under 48℃and 3h.Through optimizing the condition of target gene hybridizing, the hybridization efficiency was improved and laid a basis for further research on detection assays.3 application of shared detection microarray assay of swine diarrhea virusesThrough quality evaluation of this shared detection microarray, piglets samples with diarrhea in Sichuan province were detected by this assay.This research evaluated the specificity, sensitivity and repeatability of this gene chip through probe hybridization. The hybridization with PoRV、TGEV、PEDV、 PRRSV+CSFV+JEV+PRV suggested that the gene chip has good specificity. The sensitivity test shown that the lowest concentration for gene chip detection was 104copies/μL.5 intro-batch gene chips selected randomly were used to carry out the duplication test, and the results shown that the method had good repeatability and stability. Each gene chip can be used up to 7 times. Different RNA extraction kits were screened, and samples with detection results were detected by shared detection microarray.In this research,56 small intestine tissues with diarrhea from Mianyang, Aba, Pengzhou, Ziyang, Meishan, Guangan, Panzhihua and Bazhong were detected by shared detection microarray and RT-PCR respectively, result showed that 95% matched.22 samples of intestine contents and faeces were randomly selected and detected, whose relevance ratio was lower than that of intestine tissues. The result of detection of the 56 samples in Sichuan province showed that there were 77% positive rate in which mostly was PEDV with an infection of 66%.Through the quality evaluation of this shared detection microarray, it was showed that the assay shared detect the three diarrhea viruses with shared detection microarray was successful, and it also showed that PEDV was epidemic in piglets under 30-day-old in Sichuan province.