Rapid and Ultrasensitive Detection of Plasmodium spp. Parasites via the RPA-CRISPR/Cas12a Platform

Abstract

Microscopic examination of thick and thin blood smears stained with Giemsa dye is considered the primary diagnostic tool for the confirmation and management of suspected clinical malaria. However, detecting gametocytes is relatively insensitive, particularly in asymptomatic individuals with low-density Plasmodium infections. To complement existing diagnostic methods, a rapid and ultrasensitive point-of-care testing (POCT) platform for malaria detection is urgently needed and necessary. A platform based on recombinase polymerase amplification (RPA) followed by CRISPR/Cas12a (referred to as RPA-CRISPR/Cas12a) was developed and optimized for the determination of Plasmodium spp. parasites, particularly Plasmodium falciparum, using a fluorescence-based assay (FBDA), lateral flow test strips (LFTS), or naked eye observation (NEO). Then, the established platform was assessed with clinical malaria isolates. Under optimal conditions, the detection threshold was 1 copy/μL for the plasmid, and the limit of detection was 3.11–7.27 parasites/μL for dried blood spots. There was no cross-reactivity against blood-borne pathogens. For the accuracies of RPA-CRISPR/Cas12a, Plasmodium spp. and P. falciparum testing were 98.68 and 94.74%, respectively. The method was consistent with nested PCR results and superior to the qPCR results. RPA-CRISPR/Cas12a is a rapid, ultrasensitive, and reliable platform for malaria diagnosis. The platform requires no or minimal instrumentation for nucleic acid amplification reactions and can be read with the naked eye. Compared with similar diagnostic methods, this platform improves the reaction speed while reducing detection requirements. Therefore, this platform has the potential to become a true POCT for malaria parasites.

Design and development of a rapid meat detection system based on RPA-CRISPR/Cas12a-LFD

“…Subsequent RPA-CRISPR/Cas12a reaction conditions were as follows: RPA product (5 μL), buffer (10X, 2 μL), CrRNA (1 μM), Cas12a (Tolo Biotech, China, 0.35 μM), ssDNA fluorescent probe (0.5 μM) and nuclease-free water (3 μL). The above system was mixed and incubated at 42 °C in a fluorescence reader (Biolifesci Co., Ltd., Guangzhou, China) for 30 min, and fluorescent signals were collected every 15 s (ssDNA FQ substrate = λex: 485 nm; λem: 535 nm) ( Wei et al, 2023 ). The same RPA-CRISPR/Cas12a system was incubated for 30 min at 39 °C.…”

Detection of Parasites in the Field: The Ever-Innovating CRISPR/Cas12a

“…CrRNA can be stabilized upon binding to CRISPR/Cas12a effector proteins, especially in the lyophilized powder state, and thus can be used in combination with POC devices to form an efficient nucleic acid detector. The sensors developed to date have incorporated a range of readout mechanisms, including fluorescence [ 89 , 133 , 135 , 136 ], colorimetric [ 130 , 132 , 133 , 136 , 137 , 138 , 139 , 140 ], and electronic methods [ 104 , 141 , 142 ], ensuring system stability while improving the availability of test results. Nevertheless, achieving a balance between cost-effectiveness, quality, and convenience remains a challenge for these sensors [ 76 ].…”

“Three-in-One” Plasmonic Au@PtOs Nanocluster Driven Lateral Flow Assay for Multimodal Cancer Exosome Biosensing

“…Lateral flow assay (LFA) has attracted wide attention in the diagnostic field due to the advantages of rapidity, low cost, and ease-of-use. − These years, LFA has been applied in various fields including environmental pollutant analysis, , food safety surveillance, , and particularly point-of-care disease diagnosis. − However, traditional Au nanoparticles (Au NPs) based LFA can only provide limited sensitivity and quantitative ability; therefore, developing novel strategies to improve the sensitivity and quantitative capability of LFAs was urgently required. , …”