Ultrasensitive Colorimetric Detection of Murine Norovirus Using NanoZyme Aptasensor

Human norovirus (NoV) remains the most common cause of viral gastroenteritis and the leading cause of viral foodborne outbreaks globally. NoV is highly pathogenic with an estimated median viral infective dose (ID₅₀) ranging from 18 to 1015 genome copies. For NoV detection, the only reliable and sensitive method available for detection and quantification is reverse transcription quantitative polymerase chain reaction (RTqPCR). NoV detection in food is particularly challenging, requiring matrix specific concentration of the virus and removal of inhibitory compounds to detection assays. Hence, the RTqPCR method poses some challenges for rapid in-field or point-of-care diagnostic applications. We propose a new colorimetric NanoZyme aptasensor strategy for rapid (10 min) and ultrasensitive (calculated Limit of Detection (LoD) of 3 viruses per assay equivalent to 30 viruses/mL of sample and experimentally demonstrated LoD of 20 viruses per assay equivalent to 200 viruses/mL) detection of the infective murine norovirus (MNV), a readily cultivable surrogate for NoV. Our approach combines the enzyme-mimic catalytic activity of gold nanoparticles with high target specificity of an MNV aptamer to create sensor probes that produce a blue color in the presence of this norovirus, such that the color intensity provides the virus concentrations. Overall, our strategy offers the most sensitive detection of norovirus or a norovirus surrogate achieved to date using a biosensor approach, enabling for the first time, the detection of MNV virion corresponding to the lower end of the ID₅₀ for NoV. We further demonstrate the robustness of the norovirus NanoZyme aptasensor by testing its performance in the presence of other nontarget microorganisms, human serum and shellfish homogenate, supporting the potential of detecting norovirus in complex matrices. This new assay format can, therefore, be of significant importance as it allows ultrasensitive norovirus detection rapidly within minutes, while also offering the simplicity of use and need for nonspecialized laboratory infrastructure.