Sensitive and rapid detection of Zika virus by loop-mediated isothermal amplification

Abstract

Zika virus (ZIKV) is a mosquito-borne flavivirus, which is a pathogen affecting humans in Africa, Asia, and America. It is necessary to detect ZIKV with a rapid and sensitive molecular method to guide timely treatment. In this study, a loop-mediated isothermal amplification (LAMP) assay was described, which is an attractive option as a fast, sensitive, and specific method for ZIKV detection using the NS5 protein coding region and the envelope protein (EP) coding region as target sequences. Two different techniques, a calcein/Mn ²⁺ complex chromogenic method and real-time turbidity monitoring, were employed. The specificity and sensitivity of the LAMP assay were determined. The assay’s detection limit was 0.5 × 10 ⁻⁹ pmol/µl DNA for NS5 protein coding region and 1.12 × 10 ⁻¹¹ pmol/µl DNA for E coding region, respectively, which is a 100-fold increase in sensitivity compared with real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional PCR. All 12 non-ZIKA respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for ZIKV. In conclusion, a visual detection LAMP assay was developed, which could be a useful tool for primary quarantine purposes and clinical screening, especially in situations where resources are poor and in point-of-care tests.