Abstract：Gene chip standardization is the basic guarantee for the specific sensitive detection of pathogens, In this study, the optimal hybridization conditions of gold label silver stain visual DNA microarray such as the use of spotting buffer, spotting frequency, hybridization temperature, hybridization time, nanogold concentration and silver enhancement time were screened respectively to analysis the influence on the visual DNA microarray. Primers were designed based on the sequences of S and M gene of Porcine epidemic diarrhea virus (PEDV); S and N gene of Transmissible gastroenteritis virus (TGEV); VP7 and NSP4 gene of Group A porcine rotavirus (GAR). The 60-mer oligonucleotides probes with 15 T bases on their 5' end were designed according targets DNA sequences and sprayed on the aldehyde-modified glass slides with spotting buffer in ratio of 1∶1 and nonmiscible in once, twice and trice respectively. The positive target DNAs which were extracted from three porcine diarrhea viruses were modified biotinylated by asymmetrical PCR and hybridized in 30, 60, 90 and 120 min respectively with prepared microarray at 40, 45, 50 and 55 ℃. The nanogold particles of 10, 20, 30, 40, 50 and 60 times dilution concentration were introduced by nanogold-streptavidin particles according the specific links of biotin and streptavidin. The results could be observed by naked eyes after different minutes of silver enhancement system. To evaluated the visual DNA microarray, a total of 173 clinical samples, which collected from piglet diarrheic cases were detected using this visual DNA microarray and RT-PCR respectively. The results of 10 times of repeated tests showed that the optimal conditions of visual DNA microarray were that targets DNA hybridized with prepared microarray which is spotted once with the mixture of probes and spotting buffer in the ratio of 1∶1 at 50 ℃ in 90 min, and then enhanced by silver buffer in 12~14 min after combined with 40 μg/mL nanogold-streptavidin particles. The clinical samples results detected by microarray were consistent with that by RT-PCR. In this study, the optimal reaction conditions of viral diarrhea visual inspection were determined, which laid a foundation for the clinical application standardization research of visual microarray technology.
