Exotic and Emerging Avian Viral Disease Research Unit, Southeast Poultry Research Laboratories, US National Poultry Research Center, 934 College Station Road, Athens, GA, 30605, USA.
Sci Rep. 2024 Aug 5;14(1):18047. doi: 10.1038/s41598-024-68816-7.
Newcastle disease (ND), an economically important disease in poultry, is caused by virulent strains of the genetically diverse Orthoavulavirus javaense (OAVJ). Laboratories rely on quantitative real-time reverse transcription PCR (qRT-PCR) to detect OAVJ and differentiate between OAVJ pathotypes. This study demonstrates that a fusion cleavage site based molecular beacon with reverse transcription loop mediated isothermal amplification (MB-RT-LAMP) assay can detect and differentiate OAVJ pathotypes in a single assay. Data show that the assay can rapidly identify diverse OAVJ genotypes with sensitivity only one log-fold lower than the current fusion qRT-PCR assay (10 copies), exhibits a high degree of specificity for OAVJ, and the molecular beacon can differentiate mesogenic/velogenic sequences from lentogenic sequences. Further, data show that a two-minute rapid lysis protocol preceding MB-RT-LAMP can detect and differentiate OAVJ RNA from both spiked samples and oropharyngeal swabs without the need for RNA isolation. As the MB-RT-LAMP assay can rapidly detect and discriminate between lentogenic and mesogenic/velogenic sequences of OAVJ within one assay, without the need for RNA isolation, and is adaptable to existing veterinary diagnostic laboratory workflow without additional equipment, this assay could be a rapid primary screening tool before qRT-PCR based validation in resource limited settings.
新城疫（ND）是一种严重危害家禽的疾病，由遗传多样性的禽正黏病毒科甲型流感病毒（OAVJ）强毒株引起。实验室依赖于实时荧光定量 RT-PCR（qRT-PCR）来检测 OAVJ 并区分其不同的致病型。本研究表明，基于融合裂解位点的分子信标逆转录环介导等温扩增（MB-RT-LAMP）检测方法可在单次检测中同时检测和区分 OAVJ 致病型。研究数据显示，该检测方法能够快速识别不同的 OAVJ 基因型，其灵敏度仅比目前的融合 qRT-PCR 检测方法低一个对数（10 拷贝），对 OAVJ 具有高度特异性，且分子信标可区分中发型/强毒型序列与弱毒型序列。此外，数据还表明，在 MB-RT-LAMP 之前采用两分钟快速裂解方案，无需 RNA 提取，即可从加标样本和咽拭子中检测和区分 OAVJ RNA。由于 MB-RT-LAMP 检测方法无需 RNA 提取，即可在单次检测中快速检测和区分 OAVJ 的弱毒型和中发型/强毒型序列，并且适用于现有的兽医诊断实验室工作流程，无需额外设备，因此，在资源有限的情况下，该检测方法可作为 qRT-PCR 验证的快速初步筛选工具。
