Pan-serotype reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting 2B-NSP coding region for colorimetric detection of foot-and-mouth disease virus in clinical samples

Foot-and-mouth disease (FMD) is a highly contagious viral disease of even-toed animals. Rapid, early, and accurate diagnosis of the disease is important for the swift control of FMD. Although PCR-based assays are being used routinely for the effective diagnosis of FMD, these assays require sophisticated equipment, dedicated manpower, and complex procedures for the detection of amplified viral-genome. Colorimetric isothermal amplification assay with a sharp contrast in colour changes for the positive amplification of viral-genome would be qualified for quick and simple diagnosis of FMDV in both laboratory and field. Here, we report the development and evaluation of FMDV 2B-NSP coding region-based colorimetric RT-LAMP assay for pan-serotypic detection of viral-genome. Addition of 1 mg/ml of bovine serum albumin (BSA) as an additive, could reduce the detection time of the RT-LAMP assay from 60 to 30 min/reaction. Analytical sensitivity test showed that the RT-LAMP assay can detect up to 1000 copies of FMDV genome/reaction, simultaneously, the assay was found specific for the detection of FMDV genome as revealed on testing with serotypes O, A and Asia1 circulating in India during the last two decades. In addition, analysis of 312 clinical samples from various field outbreaks of FMDV in the country showed that RT-LAMP assay exhibited a sensitivity of 96.07%, and a specificity of 100% with an overall accuracy of 97.12%. Therefore, owing to the naked eye distinct visualization of amplified product (pink to yellow colour change), the RT-LAMP assay may facilitate rapid screening of FMD-suspected clinical samples without the use of hazardous DNA-binding dyes and simultaneously prevents aerosolization of amplified product, and subsequent carry over contamination in the diagnostic laboratory.