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 EID 50 (50% egg infective dose), except that of IBV, which was 1 EID 50 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. Electronic supplementary material The online version of this article (10.1186/s12917-019-1985-7) contains supplementary material, which is available to authorized users.

Development of a quadruple PCR-based gene microarray for detection of vaccine and wild-type classical swine fever virus, African swine fever virus and atypical porcine pestivirus

“…Recently, gene microarray has been widely used in medical science and has achieved outstanding results in the research of gene expression, pathogenesis, clinical diagnosis, drug development, and biological detection [ 14 , 15 ]. Previous studies have developed microarray assays for the simultaneous detection of avian respiratory viral diseases, including avian influenza, Newcastle disease, and infectious bronchitis virus [ 16 ]. A similar system was also established for the detection of seven cattle pathogens.…”