Genetic detection of peste des petits ruminants virus under field conditions: a step forward towards disease eradication

Abstract

BackgroundThe devastating viral disease of small ruminants namely Peste des petits ruminants (PPR) declared as target for “Global Eradication” in 2015 by the Food and Agriculture Organization (FAO) and the World Organization for Animal Health (OIE). For a successful eradication campaign, molecular diagnostic tools are preferred for their specificity, efficacy and robustness to compliment prophylactic measures and surveillance methods. However, molecular tools have a few limitations including, costly equipment, multi-step template preparation protocols, target amplification and analysis that restrict their use to the sophisticated laboratory settings. As reverse transcription-loop mediated isothermal amplification assay (RT-LAMP) has such an intrinsic potential for point of care diagnosis, this study focused on the genetic detection of causative PPR virus (PPRV) in field conditions. It involves the use of a sample buffer that can precipitate out virus envelope and capsid proteins through ammonium sulphate precipitation and exposes viral RNA, present in the clinical sample, to the LAMP reaction mixture.ResultsThe test was evaluated using 11 PPRV cultures, and a total of 46 nasal swabs (n = 32 collected in the field outbreaks, n = 14 collected from experimentally inoculated animals). The RT-LAMP was compared with the reverse transcription-PCR (RT-PCR) and real-time quantitative RT-PCR (RT-qPCR) for its relative specificity, sensitivity and robustness. RT-LAMP detected PPRV in all PPRV cultures in or less than 30 min. Its detection limit was of 0.0001TCID50 (tissue culture infective dose-50) per ml with 10-fold higher sensitivity than that of RT-PCR. In 59.4% of the field samples, RT-LAMP detected PPRV within 35–55 min. The analytical sensitivity and specificity of the RT-LAMP were equivalent to that of the RT-qPCR. The time of detection of PPRV decreased by at least forty minutes or 3–4 h in case of in the RT-LAMP as compared with the RT-qPCR and the RT-PCR, respectively.ConclusionsThe sensitive and specific RT-LAMP test developed in this study targeting a small fragment of the N gene of PPRV is a rapid, reliable and applicable molecular diagnostic test of choice under the field conditions. RT-LAMP requiring minimal training offers a very useful tool for PPR diagnosis especially during the “Global PPR Eradication Campaign”.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0940-0) contains supplementary material, which is available to authorized users.

Loop-Mediated Isothermal Amplification Assay for Peste des Petits Ruminants Virus Detection and its Comparison with RT-PCR

“…In this way, LAMP detected as little as 11.3 ng/µl of RNA which is one serial dilution higher than detected by RT-PCR i-e, 10 -3 was the last dilution that could be detected; therefore, LAMP is 10-times more sensitive in comparison to RT-PCR. This finding is in-line with Ding et al (2014) and Ashraf et al (2017). These results showed that LAMP assay is useful for detection of PPRV at low levels especially for confirmation at early stages when virus titers are relatively low (Dadas et al, 2012;Balamurugan et al, 2014).…”

“…It is an efficient protocol which yields DNA in higher quantities that is enough for confirmation by visual examination. In case of RNA viruses, the RNA template is used directly as starting material for reverse transcription together with LAMP in a single step, hence making it suitable for diagnostic purpose (Wang et al, 2011;Ashraf et al, 2017). The process is based on strand displacement reaction and the target site is amplified via stem-loop structure rapidly with accuracy, selectivity and high specificity under the isothermal conditions (Parida et al, 2006;Wang et al, 2015).…”

A Rapid, Simple, Inexpensive, and Mobile Colorimetric Assay COVID-19-LAMP for Mass On-Site Screening of COVID-19

“…Here, we report the development and evaluation of a rapid and simple one-step colorimetric reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) assay, COVID-19-LAMP, for detection of SARS-CoV-2. RT-LAMP combines reverse transcriptase, DNA polymerase, pH indictor, and six primers to amplify RNA templates [ 18 , 19 , 20 , 21 , 22 , 23 , 24 ], causing a drop in pH and, thus, a color change from pink to yellow. Since RT-LAMP involves both reverse transcription and DNA amplification at a constant temperature without the need for a PCR thermal cycler [ 25 ], it only requires simple equipment, namely benchtop centrifuges, heat blocks, and micropipettes.…”

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.…”