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使用 RT-LAMP 分子信标检测和区分低毒力和毒力 Orthoavulavirus javaense。

Detection and differentiation of low virulence and virulent Orthoavulavirus javaense using a molecular beacon with RT-LAMP.

机构信息

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.

出版信息

Sci Rep. 2024 Aug 5;14(1):18047. doi: 10.1038/s41598-024-68816-7.

Abstract

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.

摘要

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。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f74e/11300668/9b6ddbeb1525/41598_2024_68816_Fig1_HTML.jpg

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