Development and comparative assessment of RT-qPCR and duplex RT-LAMP assays for the monitoring of Aichi Virus A (AiV-A) in untreated wastewater samples.

ABSTRACT

Diverse enteric pathogens, transmitted through human and animal feces, can cause gastroenteritis. Enteric viruses, such as human Aichi virus, specifically A (AiV-A), are emerging pathogens that cause illnesses even at low doses and are spreading globally. This research developed a reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the 3CD junction and a reverse transcription colorimetric loop-mediated isothermal amplification (RT-cLAMP) duplex assay targeting junctions 2BC and 3CD of the AiV-A genome for rapid and sensitive detection of AiV-A in metropolitan and regional wastewater samples. The performance of these assays was evaluated using control materials and by analyzing wastewater samples. In serially diluted control materials, RT-qPCR provided quantifiable data (mean 1.51 log10 GC/2 µL of nucleic acid) down to a dilution of 1 × 10-5 pg/µL. In comparison, the duplex RT-cLAMP assay detected down to 1 × 10-4 pg/µL, indicating that its sensitivity was one order of magnitude less than that of RT-qPCR. Of the 38 wastewater samples from 38 metropolitan and regional wastewater treatment plants (WWTPs) in Queensland, Australia, 21 (55.3 %) tested positive by RT-qPCR with concentrations in ranging from 3.60 to 6.23 log10 GC/L. In contrast, only 15 (39.5 %) of 38 wastewater samples were positive using the duplex RT-cLAMP assay. The methods demonstrated substantial qualitative agreement (κ = 0.730), with a concordance of 86.5 %, demonstrating the reliability of RT-cLAMP for detecting AiV-A in wastewater samples. The duplex RT-cLAMP assay, despite demonstrating reduced detection sensitivity, has proven effective and holds promise as a supplementary approach, especially in settings with limited resources where rapid and affordable testing is crucial.