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A portable sequencing-based RT-LAMP approach for viral surveillance

Several recent outbreaks of mosquito-borne viral diseases such as chikungunya, zika fever, dengue fever, and malaria have been reported with severe outcomes. Differential diagnosis based on symptoms remains challenging as these viruses share the same vectors, have similar pathognomic features, and may co-circulate in overlapping geographical areas. Definitive diagnosis relies on serology, virus isolation, or genome detection from patient samples. However, these methods are expensive and require well-equipped facilities which are generally not available in remote areas. Reliable on-site diagnostic tools for the infective pathogens are urgently needed in endemic regions.

Loop-mediated isothermal amplification (LAMP) is a sensitive and robust method for nucleic acid detection that does not require expensive or sophisticated equipment and can be easily implemented in field testing. In a recent publication of PLOS Neglected Tropical Diseases, Hayashida et. al uses reverse transcription with LAMP (RT-LAMP) for the detection of the chikungunya virus (CHIKV), a positive-stranded RNA virus. The authors established an all-in-one dried RT-LAMP mix with GelGreen® as the reporter dye. The mix is stable at ambient temperatures and can reliably detect CHIKV genomes directly from clinical samples without the need for purification. Further studies show that products of the RT-LAMP reaction could be sequenced by a MinION, a portable and affordable next-generation sequencer. The study describes a simple and inexpensive point-of-care test for diagnosing CHIKV infected patients. The study also shows broader applicability for the detection and monitoring of other new and emerging viral outbreaks in remote areas.

Full citation

Hayashida K, Orba Y, Sequeira PC, Sugimoto C, Hall WW, Eshita Y, Suzuki Y, Runtuwene L, Brasil P, Calvet G, Rodrigues CDS, Santos CCD, Mares-Guia MAM, Yamagishi J, Filippis AMB, Sawa H. Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing. PLoS Negl Trop Dis. 2019 Jun 3;13(6):e0007480. doi: 10.1371/journal.pntd.0007480.

Learn more about Biotium’s safe and non-toxic nucleic acid gel stains GelGreen® and GelRed®, and qPCR dye EvaGreen®. Also see our selection of reagents for PCR and genomics including qPCR master mixes and dsDNA and RNA quantitation kits.

Reaction tubes with all-in-one dried RT-LAMP mix containing decreasing concentrations of CHIKV RNA without (upper panel) or with blood (lower panel) showing amplification based on GelGreen® fluorescence as detected by a 505 nm blue green LED detector. Credit: Hayashida et al.