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

Abstract

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 CT >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.

Eradicating the Scourge of Peste Des Petits Ruminants from the World

“…Standardization of tools should include tests for confirming outbreaks, tracking molecular epidemiology, supporting diagnostics for use in the field (pen-side test) and serological monitoring of vaccinated flocks. Assays with higher sensitivity, such as competitive ELISA, qRT-PCR and Loop Mediated Isothermal Amplification (LAMP) are clearly important for early diagnosis of PPR and also, theoretically, during the late stages of eradication or when sampling atypical hosts, e.g., wildlife with suspected infection [10]. During the latter stages of any control programme, suspected/doubtful outbreaks will also have to be reconfirmed using multiple laboratory tests [2,[8][9][10].…”

“…Assays with higher sensitivity, such as competitive ELISA, qRT-PCR and Loop Mediated Isothermal Amplification (LAMP) are clearly important for early diagnosis of PPR and also, theoretically, during the late stages of eradication or when sampling atypical hosts, e.g., wildlife with suspected infection [10]. During the latter stages of any control programme, suspected/doubtful outbreaks will also have to be reconfirmed using multiple laboratory tests [2,[8][9][10]. Defining when and how to implement an appropriate range of diagnostic tests remains a multifactorial challenge, dependent on assay availability, training needs and deficits, various cost-benefit analyses and the correct validation of the assays involved.…”

Peste des petits ruminants in large ruminants, camels and unusual hosts

“…Indeed, continuous disease surveillance, along with realistic epidemiological modelling (which needs the engagement of local communities; Fischer et al 2016), could facilitate choosing the best context-specific strategy, including vaccination frequency, spatial setting of vaccinations, target species, and quick and timely diagnosis. In this regard, either based upon immunological response or direct antigen detection, several specific and sensitive laboratory methods with rapid turn-around are available for confirmation of PPRV infection (Santhamani et al 2016;Mahapatra et al 2019). In fact, accurate and advanced disease diagnostic techniques and vaccine constructs targeting the prevalent strain are essential for effective disease control.…”

Paradigm shift in the diagnosis of peste des petits ruminants: scoping review

“…A novel inexpensive RT-LAMP provides an isothermal method to amplify viral RNA without the requirement of expensive specific thermal cycler [29,30]. Moreover, RT-LAMP reagents can be stored at ambient temperature for at least 2 weeks.…”