Sensitive and semiquantitative detection of soil-transmitted helminth infection in stool using a recombinase polymerase amplification-based assay

Abstract

Background Soil-transmitted helminths (STHs) are parasitic nematodes that inhabit the human intestine. They affect more than 1.5 billion people worldwide, causing physical and cognitive impairment in children. The global strategy to control STH infection includes periodic mass drug administration (MDA) based on the results of diagnostic testing among populations at risk, but the current microscopy method for detecting infection has diminished sensitivity as the intensity of infection decreases. Thus, improved diagnostic tools are needed to support decision-making for STH control programs. Methodology We developed a nucleic acid amplification test based on recombinase polymerase amplification (RPA) technology to detect STH in stool. We designed primers and probes for each of the four STH species, optimized the assay, and then verified its performance using clinical stool samples. Principal findings Each RPA assay was as sensitive as a real-time polymerase chain reaction (PCR) assay in detecting copies of cloned target DNA sequences. The RPA assay amplified the target in DNA extracted from human stool samples that were positive for STH based on the Kato-Katz method, with no cross-reactivity of the non-target genomic DNA. When tested with clinical stool samples from patients with infections of light, moderate, and heavy intensity, the RPA assays demonstrated performance comparable to that of real-time PCR, with better results than Kato-Katz. This new rapid, sensitive and field-deployable method for detecting STH infections can help STH control programs achieve their goals. Conclusions Semi-quantitation of target by RPA assay is possible and is comparable to real-time PCR. With proper instrumentation, RPA assays can provide robust, semi-quantification of STH DNA targets as an alternative field-deployable indicator to counts of helminth eggs for assessing infection intensity.

Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives

“…114 2.2.3 Reverse transcription-recombinase polymerase amplication (RT-RPA). RPA is considered as an alternative nucleic acid detection technique to PCR, 138 which is capable of realizing single-molecule nucleic acid detection at room temperature within 15 min without hardware equipment. 139 The addition of reverse transcriptase to the RPA reaction components enables the detection of RNA viruses in a single tube.…”

Development of real‐time recombinase polymerase amplification (RPA) and RPA combined with lateral flow dipstick (LFD) assays for the rapid and sensitive detection of cyprinid herpesvirus 3

“…Recombinase polymerase amplification‐lateral flow dipstick (RPA‐LFD) was advantageous, because it was time‐saving, sensitive, cheap, operationally simple and rapid, and can be used outside of the diagnostic lab. RPA or RPA‐LFD assay is used to rapidly detect cyprinid herpesvirus 2 (CyHV‐2) in goldfish (Preena et al., 2022), Vibrio parahaemolyticus in seafood (Geng et al., 2019), Tetracapsuloides Bryosalmonae in salmonids (Soliman et al., 2018), chilli veinal mottle virus (ChiVMV) in tobacco plants (Jiao et al., 2020), fish sex identification in Cynoglossus semilaevis (Nie et al., 2021), and soil‐transmitted helminths in human (Cantera et al., 2021).…”

Nucleic Acid Quantification with Amplicon Yield in Recombinase Polymerase Amplification

“…Indeed, it has been shown that a single molecule of target DNA can be detected with RPA within 30 min . Yet, most successfully developed RPA assays are qualitative with a positive/negative readout for target DNA, and τ d -based quantification from real-time RPA kinetic measurements has found limited success. − This partly arises due to the poor control over undesired amplification close to the ambient temperature after initiation . Similarly, a common observation with RPA reactions has been the low and variable yield of amplicons compared to PCR, though the underlying factors contributing to this remain uncharacterized.…”