Development and evaluation of a one step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP) for rapid detection of foot and mouth disease virus in India

Abstract

A simple, rapid and sensitive diagnostic assay for Foot-and-mouth disease (FMD) is required for deployment in the field. In this study, development of Reverse Transcription-Loop Mediated Isothermal Amplification (RT-LAMP) assay based on the 3D polymerase gene for specific and rapid detection FMD virus (FMDV) was carried out. The assay was optimised with viral RNA extracted from serotype O, A and Asia 1 FMDV vaccine strains, which resulted a reliable amplification at 65°C for 60 min. The amplified RT-LAMP products were identified by agarose gel electrophoresis with ethidium bromide staining or observation by naked eye for the presence of turbidity and colour change following the addition of hydroxyl naphthol blue (HNB). The specificity of the assay was demonstrated by the absence of amplification of genome extracted from other viruses or cellular origin. With respect to analytical sensitivity the developed RT-LAMP assay was found more sensitive than routinely used multiplex PCR (mPCR). Further, the assay was evaluated with RNA extracted from cell cultured isolates (n = 50), tongue epithelial samples (n = 150) and semen samples from infected bulls (n = 13). In conclusion, RT-LAMP with HNB dye was shown to be simple, specific and sensitive assay for rapid diagnosis of FMDV infection. Further, the assay has the potential for field deployment and use for rapid FMDV surveillance in India.

Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus

“…The instrumentations needed for those assays are relatively inexpensive, less complicated, less delicate and easily decontaminated or disposed of between each use. Recently, a number of RT‐LAMP assays have been developed and validated for detection and typing of FMDV (Dukes et al., ; Chen et al., ,b; Yamazaki et al., ; Ding et al., ; Kasanga et al., ; Ranjan et al., ; Waters et al., ; Farooq et al., ; Howson et al., ). The assays were rapid, highly sensitive and specific, and more resistant to inhibitors than conventional and RRT‐PCR assays.…”

“…FMD outbreaks have to be quickly detected and contained, and therefore, onsite detection tools are highly desirable. To date, a number of field‐deployable molecular assays have been developed and evaluated for their potential to use in the field to support local decision‐making (Dukes et al., ; Chen et al., ,b; Madi et al., ; Abd El Wahed et al., ; Yamazaki et al., ; Kasanga et al., ; Ranjan et al., ; Waters et al., ; Howson et al., ), and some of them have been successfully tested in the field (Madi et al., ; Abd El Wahed et al., ; Howson et al., ). To our knowledge, none of those assays are commercially available yet.…”

Defining the relative performance of isothermal assays that can be used for rapid and sensitive detection of foot-and-mouth disease virus

“…As an alternative, a number of isothermal chemistries have been adapted for detection of FMDV. To date, there are 17 FMDV-specific publications detailing four isothermal chemistries: reverse transcription loop-mediated isothermal amplification ([RT-LAMP] Dukes et al, 2006 , Li et al, 2009 , Ranjan et al, 2014 , Shao et al, 2010 , Chen et al, 2011a , Chen et al, 2011b , Yamazaki et al, 2013 , Madhanmohan et al, 2013 , Guan et al, 2013 , Waters et al, 2014 , Ding et al, 2014 , Howson et al, 2015 ), reverse transcription recombinase polymerase amplification ([RT-RPA] Abd El Wahed et al, 2013 ), nucleic acid sequence based amplification ([NASBA] Collins et al, 2002 , Lau et al, 2006 , Lau et al, 2007 ) and reverse transcription helicase dependent amplification ([RT-HDA] Jingwei et al, 2014 , Jing et al, 2013 ). Two of these (RT-LAMP and RT-RPA) have been transitioned into portable formats and successfully trialled in endemic settings ( Abd El Wahed et al, 2013 , Howson et al, 2015 ).…”

Development of a multiplex loop-mediated isothermal amplification method for the simultaneous detection of Salmonella spp. and Vibrio parahaemolyticus

“…With 4~6 specifically designed primers that target 6~8 distinct regions of the target gene, a large amount of DNA can be synthesized under a constant temperature in less than 60 min, and the amplified products can be detected by the naked eye. As a result, the LAMP assay has been widely applied in the detection of pathogenic bacteria, viruses, and parasites10111213. However, restriction enzyme analysis of amplified products and probe-based methods, which has been attempted, is laborious and complex141516.…”