MOE Joint International Research Laboratory of Animal Health and Food Safety, Institute of Immunology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.
Jiangsu Engineering Laboratory of Animal Immunology, Institute of Immunology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.
BMC Vet Res. 2019 Jul 19;15(1):253. doi: 10.1186/s12917-019-1985-7.
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.
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% egg infective dose), except that of IBV, which was 1 EID 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.
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.
禽流感病毒（AIV）、传染性支气管炎病毒（IBV）和新城疫病毒（NDV）是重要的禽类病原体，可给家禽养殖业造成巨大的经济损失。不同的呼吸道病原体可能引发相似的临床症状，导致鉴别诊断困难。重要的是，AIV 给人类公共卫生带来严重威胁。因此，迫切需要一种能够快速且同时区分这些病毒的新方法。
本研究开发了一种寡核苷酸微阵列系统。该系统可同时检测和区分 AIV（包括 H5、H7 和 H9 亚型）、NDV 和 IBV。首先设计并喷射打印了针对 AIV、NDV 和 IBV 的 3 个探针，以及针对 AIV 的 H5、H7 和 H9 亚型的另外 3 个探针，以便同步检测这 6 种病原体。标记的多重逆转录聚合酶链反应（PCR）产物与特定探针杂交，直接用肉眼读取杂交结果。该寡核苷酸微阵列检测法与其他 10 种 AIV 亚型和传染性法氏囊病病毒无交叉反应，表明该方法具有高度特异性。该方法的灵敏度至少比常规 PCR 高 100 倍，NDV、AIV、H5、H7 和 H9 的检测限可达 0.1 EID（半数鸡胚感染剂量），而 IBV 的检测限为每个反应 1 EID。在对 93 个现场样本的验证中，寡核苷酸微阵列和病毒分离法在 53 个（56.99%）样本中检测到 AIV、IBV 和 NDV，而常规 PCR 法在 50 个（53.76%）样本中检测到 AIV、IBV 和 NDV。
我们成功开发了一种使用寡核苷酸微阵列区分 AIV、NDV、IBV、H5、H7 和 H9 亚型 AIV 的方法。该微阵列是一种准确、高通量且相对简单的快速检测禽类呼吸道病毒病的方法。它可用于 AIV、IBV 和 NDV 的流行病学监测和诊断。
