Development of oligonucleotide microarray for accurate and simultaneous detection of avian respiratory viral diseases

Abstract

Background: Avian influenza virus (AIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV) are important avian pathogens that can cause enormous economic loss on the poultry industry. Different respiratory etiological agents may induce similar clinical signs that make differential diagnosis difficult. Importantly, AIV brings about severe threat to human public health. Therefore, a novel method that can distinguish these viruses quickly and simultaneously is urgently needed. Results: In this study, an oligonucleotide microarray system was developed. AIV, including H5, H7, and H9 subtypes; NDV; and IBV were simultaneously detected and differentiated on a microarray. Three probes specific for AIV, NDV, and IBV, as well as three other probes for differentiating H5, H7, and H9 of AIV, were first designed and jet-printed to predetermined locations of initiator-integrated poly(dimethylsiloxane) for the synchronous detection of the six pathogens. The marked multiplex reverse transcription polymerase chain reaction (PCR) products were hybridized with the specific probes, and the results of hybridization were read directly with the naked eyes. No cross-reaction was observed with 10 other subtypes of AIV and infectious bursal disease virus, indicating that the oligonucleotide microarray assay was highly specific. The sensitivity of the method was at least 100 times higher than that of the conventional PCR, and the detection limit of NDV, AIV, H5, H7, and H9 can reach 0.1 EID50 (50% egg infective dose), except that of IBV, which was 1 EID50 per reaction. In the validation of 93 field samples, AIV, IBV, and NDV were detected in 53 (56.99%) samples by oligonucleotide microarray and virus isolation and in 50 (53.76%) samples by conventional PCR. Conclusions: We have successfully developed an approach to differentiate AIV, NDV, IBV, H5, H7, and H9 subtypes of AIV using oligonucleotide microarray. The microarray is an accurate, high-throughput, and relatively simple method for the rapid detection of avian respiratory viral diseases. It can be used for the epidemiological surveillance and diagnosis of AIV, IBV, and NDV.