Rapid Detection of Peste des Petits Ruminants Virus (PPRV) Nucleic Acid Using a Novel Low-Cost Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for Future Use in Nascent PPR Eradication Programme
Rapid Detection of Peste des Petits Ruminants Virus (PPRV) Nucleic Acid Using a Novel Low-Cost Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for Future Use in Nascent PPR Eradication Programme
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DOI：
10.3390/v11080699
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发表时间：
2019-08-01
期刊：
影响因子：
4.7
通讯作者：
Parida, Satya
中科院分区：
文献类型：
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作者：
Mahapatra, Mana;Howson, Emma;Parida, Satya
小反刍动物疫病(PPR)是由小反刍动物疫病病毒(PPRV)引起的一种小型反刍动物疾病，流行于亚洲、中东和非洲。有效的控制包括应用早期预警系统、准确的实验室诊断和报告、限制动物行动、适当的疫苗接种和监测计划，以及通过高效的兽医服务协调所有这些措施。包括常规逆转录聚合酶链式反应(RT-PCR)和实时定量RT-PCR(RT-qPCR)在内的分子检测方法提高了PPR诊断的敏感性和快速性。然而，目前这些化验仅在实验室环境中进行；因此，针对PPR的现场诊断的开发将促进控制政策的快速执行，特别是在目标是到2030年根除PPR的情况下。环介导的等温扩增(LAMP)分析简单、快速，在RT-qPCR的范围内具有灵敏度和特异性；可以使用一次性耗材和便携式设备在现场进行。本研究建立了一种新的针对N蛋白基因的RT-LAMP方法，用于检测猪繁殖与呼吸综合征病毒的核酸。RT-LAMP检测使用的是培养的猪伪狂犬病病毒、临床感染动物的现场样本和实验感染动物的样本，这些样本涵盖了猪繁殖与呼吸综合征病毒的所有四个谱系(I-IV)。以C-T>；40为RT-qPCR临界值时，该试验与RT-qPCR的符合率为100%。此外，使用实验和暴发样本对RT-LAMP检测进行了评估，而不需要事先提取RNA，从而使其具有更多的时间和成本效益。本试验提供了一种笔侧、快速和廉价的PPR诊断方法，用于新生的PPR根除计划的现场使用。
Peste des petits ruminants (PPR) is a disease of small ruminants caused by peste des petits ruminants virus (PPRV), and is endemic in Asia, the Middle East and Africa. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Molecular assays, including conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR) have improved the sensitivity and rapidity of diagnosing PPR. However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for PPR would improve the fast implementation of control policies, particularly when PPR has been targeted to be eradicated by 2030. Loop-mediated isothermal amplification (LAMP) assays are simple to use, rapid, and have sensitivity and specificity within the range of RT-qPCR; and can be performed in the field using disposable consumables and portable equipment. This study describes the development of a novel RT-LAMP assay for the detection of PPRV nucleic acid by targeting the N-protein gene. The RT-LAMP assay was evaluated using cell culture propagated PPRVs, field samples from clinically infected animals and samples from experimentally infected animals encompassing all four lineages (I-IV) of PPRV. The test displayed 100% concordance with RT-qPCR when considering an RT-qPCR cut-off value of C-T >40. Further, the RT-LAMP assay was evaluated using experimental and outbreak samples without prior RNA extraction making it more time and cost-effective. This assay provides a solution for a pen-side, rapid and inexpensive PPR diagnostic for use in the field in nascent PPR eradication programme.