Sandwich hybridization-based loop-mediated isothermal amplification (SHB-LAMP) for high-throughput detection of malaria RNA from asymptomatic infections

Abstract

To prevent transmission of Plasmodium parasite and finally eliminate malaria, mass screening of asymptomatic infections in low malaria-endemic regions is often required but remains a challenge. Traditional molecular diagnostic strategies require operating conditions that are incompatible with the low-resource settings in endemic regions, and yield results with either low throughput or limited sensitivity. We developed high-throughput, sensitive, rapid, cost-effective and user-friendly sandwich hybridization-based LAMP (SHB-LAMP) for the detection of RNA targets without RNA extraction or reverse transcription. RNA targets in samples are released through cell lysis and captured by a series of specific, tailed probes onto the wells of 96-well plates through sandwich hybridization reaction within 30 min. After removal of extra probes and impurities, bound loop-tailed probes are ligated to form single stranded templates for subsequent LAMP amplification and detection. Results can be read by naked eyes under a handheld UV flashlight. SHB-LAMP for Plasmodium 18 S rRNA detection provides a limit of detection better than the most sensitive LAMP- or PCR-based assays published. It uses a portable shaking heat-block and allows the test of 96 samples within 90 min. In an assay of 94 blood samples from malaria endemic areas, SHB-LAMP detected 28 more positive asymptomatic samples than standard qPCR. Thus, SHB-LAMP offers an attractive approach for large scale molecular screening of infectious diseases.