Development of real-time and lateral flow strip reverse transcription recombinase polymerase Amplification assays for rapid detection of peste des petits ruminants virus

Abstract

BackgroundPeste des petits ruminants (PPR) is an economically important, Office International des Epizooties (OIE) notifiable, transboundary viral disease of small ruminants such as sheep and goat. PPR virus (PPRV), a negative-sense single-stranded RNA virus, is the causal agent of PPR. Therefore, sensitive, specific and rapid diagnostic assay for the detection of PPRV are necessary to accurately and promptly diagnose suspected case of PPR.MethodsIn this study, reverse transcription recombinase polymerase amplification assays using real-time fluorescent detection (real-time RT-RPA assay) and lateral flow strip detection (LFS RT-RPA assay) were developed targeting the N gene of PPRV.ResultsThe sensitivity of the developed real-time RT-RPA assay was as low as 100 copies per reaction within 7 min at 40 °C with 95% reliability; while the sensitivity of the developed LFS RT-RPA assay was as low as 150 copies per reaction at 39 °C in less than 25 min. In both assays, there were no cross-reactions with sheep and goat pox viruses, foot-and-mouth disease virus and Orf virus.ConclusionsThese features make RPA assay promising candidates either in field use or as a point of care diagnostic technique.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-017-0688-6) contains supplementary material, which is available to authorized users.

Evaluation of an incubation instrument-free reverse transcription recombinase polymerase amplification assay for rapid and point-of-need detection of canine distemper virus

“…Most of the established LFS RPA assays either developed for DNA or performed the reactions in water baths or incubator block (Lillis et al, 2014;Wang et al, 2017b;Wu et al, 2017;Yang et al, 2017Yang et al, , 2016. In the LFS RT-RPA assays for PPRV and BEFV, the viral RNA was not used as the template directly, while there was an additional process to reverse transcribe the extracted RNA to cDNA prior to performing the assays, which need approximately 30-60 min (Hou et al, 2017;Yang et al, 2017). In this assay, we added MMLV (4U/μL) and RNase inhibitor (0.8U/μL) into the TwistAmp™ nfo reaction system, and the CDV LFS RT-RPA worked well with CDV RNA as the template, which is the other advantage of our assay.…”

“…Our laboratory had developed a real-time RT-RPA assay based on exo probe for real-time detection of CDV while the assay still depended on the specialized instrument, Genie III (OptiGene, West Sussex, UK) (Wang et al, 2017a). Series of LFS RPA assays had been developed for the detection of porcine parvovirus (PPV), peste des petits ruminants virus (PPRV) and bovine ephemeral fever virus (BEFV) (Hou et al, 2017;Yang et al, 2017Yang et al, , 2016.…”

Point‐of‐care diagnostic assay for rapid detection of porcine deltacoronavirus using the recombinase polymerase amplification method

“…The isothermal amplification of the RPA assay depends on three enzymes: a recombinase polymerase, a single‐stranded binding protein and a DNA polymerase (Piepenburg, Williams, Stemple, & Armes, ). RPA amplicons can be detected by gel electrophoresis or directly visualized by lateral flow dipstick (LFD) (Yang, Qin, Song, et al., ); real‐time detection of the amplification can be achieved by adding a probe to the reaction (Ma, Zeng, Huang, et al., ).…”

Equipment-free recombinase polymerase amplification assay using body heat for visual and rapid point-of-need detection of canine parvovirus 2

“…Normal human body temperature (36.1-37°C) is within the above temperature range, and several RPA assays have been developed to perform the reaction using body heat by either holding the reaction tube in the axilla or in closed fists [27,28]. Most of the established LFS RPA assays were performed by running the reactions in water baths or heating blocks [20][21][22]25,28]. In this study, the CPV-2 LFS RPA assay was performed by holding the reaction tubes in a closed fist, which is the feature of the assay.…”

“…The amplicons are then detected by the naked eye in a 'sandwich' assay format, such as a lateral flow strip (LFS), which uses anti-FAM gold conjugates and biotin-ligand molecules. A series of LFS RPA assays had been developed for the detection of porcine parvovirus (PPV), peste des petits ruminants virus (PPRV) and bovine ephemeral fever virus (BEFV) [20][21][22].…”