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

Abstract

Background: Peste 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.

Methods: In 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.

Results: The 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.

Conclusions: These features make RPA assay promising candidates either in field use or as a point of care diagnostic technique.

Keywords: Lateral flow strip; PPRV; RT-RPA; Reverse transcription recombinase polymerase amplification assay; Small ruminants.

Fig. 1

a Typical raw fluorescence data of real-time RT-RPA assay for standard RNA in vitro transcribed from the pPPRV/RPA plasmid as they are shown in the figure. NC represent negative control. b Reproducibility of the real-time RT-RPA assay. The threshold time is represented as mean ± standard deviation (SD). The standard regression line was generated based on 6 data sets (c) Probit regression analysis using Statistics software (GraphPad Prism 5) was done on data from the six runs of real-time RT-RPA assay. The limit of detection at 95% probability is depicted by a triangle

Fig. 2

Comparison between performances of PPRV real-time RT-RPA assay and real-time RT-PCR assay on positive samples (n = 14, marked by balls) and samples of spiked tissues lysates (n = 18, marked by diamonds). Linear regression analysis of the real-time RT-RPA threshold time (y axis) and RT-qPCR cycle threshold (Cq) values (x axis) were determined by Excel software

Fig. 3

Determination of reaction temperature and time. a The LFS RT-RPA assay were performed respectively at different temperatures as they are shown in the figure. b After 10 min of isothermal amplification reaction, the slight test line is visible on the test strip, and when the amplification time is longer than 15 min or 20 min, the clear test line is visible on the test strip. Including the incubation time of 2 min, the whole assay time of the LFS RT-RPA assay is less than 25 min, and the results showed that this assay works effectively in a broad range of temperatures from 37 to 45 °C

Fig. 4

a The sensitivity of LFS RT-RPA was 150 copies of the standard plasmids transcribed RNA, b and all the LFS RT-RPA positive result could be consolidated on stained agarose gel (2%) assay

Fig 5

Specificity test results of LFS RT-RPA assay using total DNA/RNA extracted from PPR virus and other virus. PPRV: peste des petits ruminants, ORFV: Orf virus, GPV: goat pox viruses, SPV: sheep pox viruses, FMDV: foot-and-mouth disease virus. a Positive RPA nfo reaction products can be detect in the lateral flow strips format (LFS RT-RPA). b Positive RPA nfo reaction products (121 bp) also can be detect on a stained agarose gel (2%)