One-step real-time loop-mediated isothermal amplification (RT-LAMP): evaluation and its application for the detection of foot-and-mouth-disease virus and its serotypes

Abstract

Availability of a rapid and sensitive diagnostic technique is key in successful prevention and control of infectious diseases like foot-and-mouth disease (FMD). Existing conventional diagnostic tests for FMD are laborious and time-consuming with low sensitivity and specificity. Molecular-based techniques are costly and difficult, involving refined apparatus like a thermal cycler. In the present study, the technique of real-time loop-mediated isothermal amplification (RT-LAMP) was standardized for diagnosing FMDV and its serotypes, evaluated using field samples, and compared with the existing real-time PCR in Pakistan. RT-LAMP amplified the target 3D gene using specific primers at 65 °C for 60 min and the VP1 gene using serotype specific primers at 63 °C for 60 min. A total of 38 samples out of 50 were positive by RT-LAMP and identified serotypes were A (n = 15), O (n = 15), and Asia-1 (n = 8). The efficiency of RT-LAMP in this study was highest for serotype Asia-1 (80.9%) followed by serotype A (73.4%) and O (62.35).

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

“…LAMP primers were also tried with IBDV and FMDV RNA samples, and there was no cross activity and primers were specific for PPRV only (Li et al, 2010;Maryam et al, 2017). These results indicate that LAMP assay is a specific technique for PPRV.…”

Advances in the Diagnosis of Foot-and-Mouth Disease

“…To date, real-time RT-LAMP (qRT-LAMP) is available for the detection and differentiation of FMDV serotypes O, A, and Asia 1 ( 47 ). Unlike RT-qPCR, qRT-LAMP for FMDV detection could not produce accurate quantitative results, as measurements of magnesium pyrophosphate by turbidity or DNA by fluorogenic dye are directly proportional to the size of LAMP products.…”

“…Thus, alternatives such as isothermal assays and dipsticks assays (also known as chromatographic strip tests) could serve as promising diagnostic methods in the field for a prompt FMD detection to allow timely implemented control measures. Reverse transcription-RPA ( 14 , 40 ), RT-LAMP ( 23 , 25 , 29 , 46 , 47 ), and nucleic-acid sequence-based amplification ( 142 ) have been used to detect FMDV. A combination of dipsticks assays with RT-LAMP and RT-RPA has also been used for virus serotyping in field samples ( 14 , 28 , 46 ).…”

Further development of a reverse‐transcription loop‐mediated isothermal amplification (RT‐LAMP) assay for the detection of foot‐and‐mouth disease virus and validation in the field with use of an internal positive control

“…This pan‐serotypic FMD RT‐LAMP assay has been further adapted for use in the field, initially being combined with a lateral flow device to detect FMDV in unextracted epithelial suspensions by simply diluting the sample (Waters et al., 2014) and then with lyophilized reagents and portable equipment in the field in Africa (Howson, Armson, et al, 2017). Despite several other LAMP assays being developed for FMD, including those for serotyping FMDV (Chen, Zhang, Liu, & Liu, 2011a; Madhanmohan et al., 2013), their limitations include an inability to amplify or lack of testing on all seven serotypes (Chen, Zhang, Liu, & Liu, 2011b; Lim et al., 2018; Maryam et al., 2017; Ranjan et al., 2014; Shao et al., 2010) or a lengthy testing procedure that is not as practical for in‐field testing (Guan et al., 2013).…”