TY - JOUR
T1 - Portable, single nucleotide polymorphism-specific duplex assay for virus surveillance in wastewater
AU - Oh, Chamteut
AU - Xun, Guanhua
AU - Lane, Stephan Thomas
AU - Petrov, Vassily Andrew
AU - Zhao, Huimin
AU - Nguyen, Thanh H.
N1 - We acknowledge funding support from the EPA grant ( R840487 ). This study has not been formally reviewed by EPA. The views expressed in this document are solely those of Professor Thanh H. Nguyen and do not necessarily reflect those of the Agency. EPA does not endorse any products or commercial services mentioned in this publication. We also thank Hankeun Lee for his advice on a portable fluorimeter.
PY - 2024/2/20
Y1 - 2024/2/20
N2 - The Argonaute protein from the archaeon Pyrococcus furiosus (PfAgo) is a DNA-guided nuclease that targets DNA with any sequence. We designed a virus detection assay in which the PfAgo enzyme cleaves the reporter probe, thus generating fluorescent signals when amplicons from a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay contain target sequences. We confirmed that the RT-LAMP-PfAgo assay for the SARS-CoV-2 Delta variant produced significantly higher fluorescent signals (p < 0.001) when a single nucleotide polymorphism (SNP), exclusive to the Delta variant, was present, compared to the samples without the SNP. Additionally, the duplex assay for Pepper mild mottle virus (PMMOV) and SARS-CoV-2 detection produced specific fluorescent signals (FAM or ROX) only when the corresponding sequences were present. Furthermore, the RT-LAMP-PfAgo assay does not require dilution to reduce the impact of environmental inhibitors. The limit of detection of the PMMOV assay, determined with 30 wastewater samples, was 28 gc/μL, with a 95 % confidence interval of [11,103]. Finally, using a point-of-use device, the RT-LAMP-PfAgo assay successfully detected PMMOV in wastewater samples. Based on our findings, we conclude that the RT-LAMP-PfAgo assay can be used as a portable, SNP-specific duplex assay, which will significantly improve virus surveillance in wastewater.
AB - The Argonaute protein from the archaeon Pyrococcus furiosus (PfAgo) is a DNA-guided nuclease that targets DNA with any sequence. We designed a virus detection assay in which the PfAgo enzyme cleaves the reporter probe, thus generating fluorescent signals when amplicons from a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay contain target sequences. We confirmed that the RT-LAMP-PfAgo assay for the SARS-CoV-2 Delta variant produced significantly higher fluorescent signals (p < 0.001) when a single nucleotide polymorphism (SNP), exclusive to the Delta variant, was present, compared to the samples without the SNP. Additionally, the duplex assay for Pepper mild mottle virus (PMMOV) and SARS-CoV-2 detection produced specific fluorescent signals (FAM or ROX) only when the corresponding sequences were present. Furthermore, the RT-LAMP-PfAgo assay does not require dilution to reduce the impact of environmental inhibitors. The limit of detection of the PMMOV assay, determined with 30 wastewater samples, was 28 gc/μL, with a 95 % confidence interval of [11,103]. Finally, using a point-of-use device, the RT-LAMP-PfAgo assay successfully detected PMMOV in wastewater samples. Based on our findings, we conclude that the RT-LAMP-PfAgo assay can be used as a portable, SNP-specific duplex assay, which will significantly improve virus surveillance in wastewater.
KW - Diagnostic assay
KW - PfAgo
KW - Portable
KW - Single nucleotide polymorphism detection
KW - Virus
KW - Wastewater
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U2 - 10.1016/j.scitotenv.2023.168701
DO - 10.1016/j.scitotenv.2023.168701
M3 - Article
C2 - 37992833
AN - SCOPUS:85178116640
SN - 0048-9697
VL - 912
JO - Science of the Total Environment
JF - Science of the Total Environment
M1 - 168701
ER -