Development of a reverse transcription loop-mediated isothermal amplification assay for rapid detection of foot-and-mouth disease virus

Abstract

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease caused by foot-andmouth disease virus (FMDV), which infects cloven-hoofed animals resulting in severe economic losses. Because very early detection of the virus is considered a cornerstone for minimization of the spread of the disease, the development of sensitive on-site testing techniques is urgently needed. Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can be performed using basic equipment and its results can be readily observed. Thus it can be used as a rapid and sensitive on-site testing technique. The aim of this study was to develop an RT-LAMP assay as a potential on-site testing technique for the rapid detection of FMDV. Evaluation of LAMP additives to enhance the specificity of the reaction to allow for reliable diagnosis was performed; the optimized RT-LAMP assay using 1.5M formamide/N,Ndimethylformamide additives and 6mM MgSO4 was shown to detect FMDV RNA using the enhanced reverse transcriptase and strand displacement activities of the Bst 3.0 DNA polymerase in less than 1 hour. SYBR Green I dye was used for detection of amplification products and the results were further validated by agarose gel electrophoresis and turbidity observation. As compared to conventional PCR, the RT-LAMP assay was ten times more sensitive when using 10-fold serially diluted cDNA as a target, and while the intensity of bands in PCR became successively fainter as the target was diluted, the SYBR Green I detection of LAMP products was uniform and unambiguous over the complete range of detection making this assay a good candidate for point of care testing and screening. The LAMP assay showed a 90% detection rate, compared to 69% for PCR, when tested on cDNA prepared from 29 samples taken from animals with clinical signs of FMDV from 2014-2018, which is another indication of the greater sensitivity of the developed RT-LAMP assay. Furthermore, all positive samples detected by PCR were also detected by RT-LAMP. These results suggest a potential use of the visually inspected RT-LAMP as a more rapid and sensitive tool for simple diagnosis of FMDV. IV