Development and application of a visual microarray for synchronously detecting H5N1, H7N9 and H9N2 avian influenza virus RNA

The aim of this study was to develop a microarray assay for the simultaneous detection of the H5, H7, H9, N1, N9 and N2 genes of the avian influenza virus (AIV) using a Nanogold-streptavidin and silver-stain-enhanced nucleic acid dot-blot hybridisation system. The conserved sequences of H5 genes from H5N1, H7 genes from H7N9, H9 genes from H9N2, N9 genes from H7N9 and N2 genes from H9N2 AIV were cloned, together with that of N1 obtained commercially, and were used as templates for generating the probes using biotin-labeled primers, which targeted the conserved regions of H5, H7, H9, N1, N9 and N2 genes, respectively. The oligonucleotide probes were diluted using the spotting buffer and ddH2O, and each probe was then spotted to each specific position on the microarray. The PCR products including biotin-labeled lambda, NP, H5, H7, H9, N1, N9 and N2 were mixed, 200 μL of which was then added to the microarray chamber after denaturing. Following a hybridization incubation at 45℃ for 120 min, the microarray was then incubated with nanogold-streptavidin about 4 μg/mL for 30 min. After the supplementary of 200 μL of silver buffer A and silver buffer B in the chamber, the hybridization results were assessed by direct visualization in the dark at room temperature. The microarray assay was optimized and its specificity, sensitivity and stability were evaluated. The optimal conditions comprised a probe concentration of 50 μmol/L, a hybridization temperature of 45℃ and a hybridization time of 2 h. The optimal concentration of nanogold-streptavidin was 4 μg/mL and the optimal staining time was 7 min. The results of specificity evaluation showed that no cross-binding of the probes with each other and no cross-hybridization with Newcastle disease virus, infectious bronchitis virus and infectious laryngotracheitis virus was observed. The optimized microarray assay was significantly more sensitivity than the reverse-transcription PCR assay. The microarray was available after storing at less 90 d at 4 ℃. The optimized microarray assay was validated on clinical specimens and the results showed that it had over 95.6 % correlation with reverse-transcription PCR method. Therefore, the microarray assay could be used for the high throughput detection of AIV infections due to H5N1, H7N9 and H9N2.