Rapid Visual Detection of Plasmodium Using Recombinase-Aided Amplification With Lateral Flow Dipstick Assay

Abstract

BackgroundMalaria is a global public health problem. China has had no case of indigenous malaria since 2016. However, imported cases of malaria remain an issue among travelers, overseas workers, and foreign traders. Although these cases are always asymptomatic, if they donate blood, there is a great risk of transfusion transmitted-malaria (TTM). Therefore, blood banks need a rapid screening tool to detect Plasmodium species.MethodsWe designed an assay using recombinase-aided amplification (RAA) and a lateral-flow dipstick (LFD) (RAA-LFD) to detect the 18S ribosomal RNA gene of Plasmodium species. Sensitivity was evaluated using a recombinant plasmid and Plasmodium genomic DNA. Specificity was evaluated using DNA extracted from the blood of patients with malaria or other infectious parasites. For clinical assessment, blood samples from patients with malaria and blood donors were evaluated.ResultsThe RAA-LFD assay was performed in an incubator block at 37°C for 15 min, and the amplicons were visible to the naked eye on the flow dipsticks within 3 min. The sensitivity was 1 copy/μL of recombinant plasmid. For genomic DNA from whole blood of malaria patients infected with P. falciparum, P. vivax, P. ovale, and P. malariae, the sensitivity was 0.1 pg/μL, 10 pg/μL, 10-100 pg/μL, and 100pg/μL, respectively. The sensitivity of this assay was 100pg/μL. No cross-reaction with other transfusion-transmissible parasites was detected.ConclusionsThe results demonstrated that this RAA-LFD assay was suitable for reliable field detection of Plasmodium species in low-resource settings with limited laboratory capabilities.

Lateral Flow Assay Sensitivity and Signal Enhancement via Laser µ-Machined Constrains in Nitrocellulose Membrane

“…Currently, LFAs serve as the most common PoC sensors for diagnosing viruses (human immunodeficiency virus (HIV), hepatitis, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [3]), bacteria (streptococcus, E. coli), and parasites (malaria [4]) caused infections. They are widely used for determining medical conditions (pregnancy testing and cardiac markers), environmental monitoring (pesticides and heavy metals [5]), food safety applications (such as allergens and pathogens [6]), and cancer markers [7] (prostate-specific antigens, alpha-fetoproteins [8]).…”

Rapid detection of multiple resistance genes to last-resort antibiotics in Enterobacteriaceae pathogens by recombinase polymerase amplification combined with lateral flow dipstick

“…Detection of RPA products is mainly completed via probe-based florescence ( Kim and Lee, 2016 ), gel electrophoresis ( Cao et al, 2018 ), or visualization by nucleic acid lateral flow dipstick (LFD) immunoassays ( Xu et al, 2018 ). Among these, LFD immunoassays are simple, fast (~5 min), cost-effective and accurate for detection of amplified products and are more suitable for point-of-care testing ( Lin et al, 2022 ). Additionally, multiplex LFD immunoassays that analyze multiple targets simultaneously have emerged in several research fields.…”

Development of Enzymatic Recombinase Amplification Assays for the Rapid Visual Detection of HPV16/18

“…Agarose gel electrophoresis can be applied to analyze the length and amounts of amplicons, but it takes a relatively long reaction time and requires other equipment [ 26 ]. In contrast, the fluorescent probe can monitor and analyze the results in real-time [ 27 - 29 ], while LFD is extremely portable and easy to operate and can read the results within 5min by the naked eye [ 30 - 32 ]. The probes and enzymes applied for the above methods are quite different.…”

“…One of the markers can be captured by the test line of LFD and the other will be combined with a gold-labeled antibody. The product of ERA-LFD assay will finally be presented as the bands on the test line [ 31 , 32 ]. The amplification process of the ERA technique is shown in Fig.…”