JEV-nanobarcode and colorimetric reverse transcription loop-mediated isothermal amplification (cRT-LAMP)

Abstract

Nucleic acid amplification tests (NAATs) are powerful tools for the Japanese encephalitis virus (JEV). We demonstrated highly sensitive, specific, and rapid detection of JEV by colorimetric reverse-transcription loop-mediated isothermal amplification (cRT-LAMP). Under optimized conditions, the RT-LAMP assay results showed that the limit of detection was approximately equivalent to 1 RNA genome copy/μL with an assay time of 30 min. The assay was highly specific to JEV when tested with other mosquito-borne virus panels (Zika virus and dengue virus types 2-4). The ability to detect JEV directly from crude human sample matrices (serum and urine) demonstrated the suitability of our JEV RT-LAMP for widespread clinical application. The JEV RT-LAMP provides combination of rapid colorimetric determination of true-positive JEV RT-LAMP amplicons with our recently developed JEV-nanobarcodes, measured at absorbance wavelenght of 530 (A₅₃₀) and 650 (A₆₅₀), which have a limit of detection of 23.3 ng/μL. The AuNP:polyA₁₀-JEV RT-LAMP nanobarcodes exhibited superior capability for stabilizing the true-positive JEV RT-LAMP amplicons against salt-induced AuNP aggregation, which improved the evaluation of true/false positive signals in the assay. These advances enable to expand the use of RT-LAMP for point-of-care tests, which will greatly bolster JEV clinical programs. The JEV RT-LAMP nanobarcode assay targeting the envelope (E) gene and MgSO₄ induced AuNP aggregation, indicated by an instant pink-to-violet colorimetric read-out.

Keywords: Absorption measurement; Colorimetric reverse transcription loop-mediated isothermal amplification (cRT-LAMP); Gold nanoparticles; Japanese encephalitis virus (JEV); Poly adenine (A10) functionalized JEV specific nanobarcode.