A Rapid On-Site Detection Method for Plasmodium falciparum Carried by Mosquitoes Using Disc Microfluidic Isothermal Amplification.

Abstract

Rapidly identifying Anopheles-carrying malaria parasites is crucial for imported malaria prevention. However, suitable methods still lack quick detection in limited-resource situations. In this study, disc microfluidic isothermal amplification integrating loop-mediated isothermal amplification (LAMP) and microfluidic chip technology were applied to develop rapid and precise detection with low resource requirements. Primer set EMP1G2, which is specific to Plasmodium falciparum (P. falciparum) erythrocyte membrane protein 1, and primer set 18sG2, which is specific to ribosomal 18s subunit RNA, were screened for optimal LAMP-specific primer sets. The minimum detection limits were 125 copies/µL for the EMP1G2 and 6,562 copies/µL for the 18sG2. Subsequently, optimal primer sets were evaluated for specificity with nucleic acid from other mosquito-borne pathogens and arthropod vectors. No nonspecific amplification was observed in optimal amplification-specific primer sets with the DNA of Anopheles mosquitoes and morphologically similar arthropods or with the copy DNA of Zika virus, yellow fever virus, or dengue virus 1. The detection method was evaluated in a simulated scenario and demonstrated a robust capacity for rapid on-site detection. Additionally, polymerase chain reaction (PCR) and quantitative real-time PCR methods were compared using this method. In this study, a rapid detection method based on disc microfluidic isothermal amplification was developed that could be used to detect P. falciparum carried by mosquitoes in a field setting under limited resource conditions.