Development of a reverse transcription loop-mediated isothermal amplification assay for the detection of vesicular stomatitis New Jersey virus: Use of rapid molecular assays to differentiate between vesicular disease viruses

Abstract

Vesicular stomatitis (VS) is endemic in Central America and northern regions of South America, where sporadic outbreaks in cattle and pigs can cause clinical signs that are similar to foot-and-mouth disease (FMD). There is therefore a pressing need for rapid, sensitive and specific differential diagnostic assays that are suitable for decision making in the field. RT-LAMP assays have been developed for vesicular diseases such as FMD and swine vesicular disease (SVD) but there is currently no RT-LAMP assay that can detect VS virus (VSV), nor are there any multiplex RT-LAMP assays which permit rapid discrimination between these 'look-a-like' diseases in situ. This study describes the development of a novel RT-LAMP assay for the detection of VSV focusing on the New Jersey (VSNJ) serotype, which has caused most of the recent VS cases in the Americas. This RT-LAMP assay was combined in a multiplex format combining molecular lateral-flow devices for the discrimination between FMD and VS. This assay was able to detect representative VSNJV's and the limit of detection of the singleplex and multiplex VSNJV RT-LAMP assays were equivalent to laboratory based real-time RT-PCR assays. A similar multiplex RT-LAMP assay was developed to discriminate between FMDV and SVDV, showing that FMDV, SVDV and VSNJV could be reliably detected within epithelial suspensions without the need for prior RNA extraction, providing an approach that could be used as the basis for a rapid and low cost assay for differentiation of FMD from other vesicular diseases in the field.

Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus

“…Therefore, if the fluorescent primer causes non-specific extension at the 3′ end, unexpected signals will be detected. To avoid non-specific signals, melting curve analysis of the LAMP amplicon is useful to confirm amplification of the targeted sequence (Fowler et al, 2016;Kurosaki et al, 2017). However, melting curve analysis requires incubation of at a higher temperature than that LAMP, and requires additional time after amplification, which negates the main advantage of LAMP.…”

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of African Horse Sickness Virus

“…() and Fowler et al. (). It would be appropriate to extend the development of this RT‐LAMP assay to introduce the aforementioned modifications and testing the performance of the assay directly in the field during real outbreak investigations.…”

“…To facilitate the use of this assay in the field, future work should consider the removal of nucleic acid extraction steps, as described by Waters et al (2014) and Howson et al (2017); the lyophilization of the assay reagents, as shown by Howson et al (2017); and the development of an alternative visualization strategy as described by Waters et al (2014), Howson et al (2017) and Fowler et al (2016). It would be appropriate to extend the development of this RT-LAMP assay to introduce the aforementioned modifications and testing the performance of the assay directly in the field during real outbreak investigations.…”

“…Loop-mediated isothermal amplification (LAMP) is an isothermal, autocycling, strand-displacement DNA amplification technique (Notomi et al, 2000) which can combine a reverse-transcription step (RT-LAMP), be performed at a single temperature in a water bath and can be combined with simple, disposable visualization using molecular lateral-flow devices (LFD's) (Waters et al, 2014;Howson et al, 2017;Fowler et al, 2016). Numerous LAMP assays have been developed for both human and veterinary pathogens including orbiviruses such as bluetongue (Mulholland et al, 2014;Mohandas et al, 2015;Maan et al, 2016), however, as yet there has not been one developed for detection of AHSV.…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…The sample of interest, for example, saliva, blood, urine and other body fluids, is applied at one end of the strip (sample pad), which is conjugated with LAMP buffer mix and biotin, making the loaded sample suitable for interaction with the detection system. Most generally, lateral flow test was established using fluorescein in isothiocyanate (FITC) labelled DNA probes (which will recognize the specific region in the LAMP amplicon) hybridized with biotinylated LAMP amplicons (Thongkao et al 2015;Fowler et al 2016;Huang et al 2017;Park et al 2017). This complex would be captured by streptavidin on the biotin, forming another complex with anti-FITC antibody coated on the gold nanoparticles.…”