Abstract
Rapid, field-based diagnostic assays are desirable tools for the control of foot-and-mouth disease (FMD). Current approaches involve either; 1) Detection of FMD virus (FMDV) with immuochromatographic antigen lateral flow devices (LFD), which have relatively low analytical sensitivity, or 2) portable RT-qPCR that has high analytical sensitivity but is expensive. Loop-mediated isothermal amplification (LAMP) may provide a platform upon which to develop field based assays without these drawbacks. The objective of this study was to modify an FMDV-specific reverse transcription–LAMP (RT-LAMP) assay to enable detection of dual-labelled LAMP products with an LFD, and to evaluate simple sample processing protocols without nucleic acid extraction. The limit of detection of this assay was demonstrated to be equivalent to that of a laboratory based real-time RT-qPCR assay and to have a 10,000 fold higher analytical sensitivity than the FMDV-specific antigen LFD currently used in the field. Importantly, this study demonstrated that FMDV RNA could be detected from epithelial suspensions without the need for prior RNA extraction, utilising a rudimentary heat source for amplification. Once optimised, this RT-LAMP-LFD protocol was able to detect multiple serotypes from field epithelial samples, in addition to detecting FMDV in the air surrounding infected cattle, pigs and sheep, including pre-clinical detection. This study describes the development and evaluation of an assay format, which may be used as a future basis for rapid and low cost detection of FMDV. In addition it provides providing “proof of concept” for the future use of LAMP assays to tackle other challenging diagnostic scenarios encompassing veterinary and human health.
Highlights
Incursions of foot-and-mouth disease (FMD) into countries or zones with FMD-free status has devastating impacts
Agarose gel electrophoresis (AGE) end-point examination of RT-Loop-mediated isothermal AMPlification (LAMP) products agreed with these results generated on a real-time PCR machine with PicoGreen as an indicator of amplified LAMP products (Figure 1c)
Biotin and fluorescein labelling of the oligos used in the Reverse transcription LAMP (RT-LAMP) assay had no effect on the limit of detection of the assay when the reactions were analysed by both agarose gel electrophoresis (AGE) and the lateral flow device (LFD) (Figure 1d)
Summary
Incursions of foot-and-mouth disease (FMD) into countries or zones with FMD-free status has devastating impacts. As a result of the economic impact in the event of an incursion of FMD, the primary goal is to return to FMD free status as quickly as possible after the first case is identified to minimise these impacts upon national livestock industries. Samples from suspected outbreaks can be tested for the presence of virus by (i) RT-qPCR [2], (ii) FMDV antigen ELISA [3], and (iii) virus isolation [4] Some of these tests are rapid, they all rely upon the transport of samples from suspect cases to centralised laboratories which can add a significant time delay from sample collection to arrival at the laboratory. The widespread dissemination of FMD in the UK in 2001 has been largely attributed to the silent spread in sheep [6], which would render field based assays reliant upon clinical signs useless in this species as sheep are well documented to often show minimal to no clinical signs associated with FMDV infection [7]
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