Use of Aptamer Magnetic Capture and Quantitative Real-time PCR (AMC-RT-qPCR) for Detection of Human Norovirus in a Model Food

Introduction: Aptamers are single-stranded (ss) DNA or RNA molecules that naturally fold into complex three-dimensional shapes with binding specificity to target molecules. Interest has emerged in their use for selective concentration and purification of pathogens from complex samples.  In previous work, we developed (ss) DNA aptamers with binding specificity to human noroviruses (HuNoV), the leading cause of foodborne illness. Two aptamer candidates (designated AP 25 and M6-2) showed broad reactivity in binding assays applied to a panel of genogroup I and II HuNoV strains.  However, the efficacy of these ligands in capture and detection of HuNoV in foods is unknown. 

Purpose: To investigate the performance of aptamers AP25 and M6-2 for detection of HuNoV in a model food system (lettuce) using Aptamer Magnetic Capture in conjunction with RT-qPCR (AMC-RT-qPCR). 

Methods: Stool samples corresponding to HuNoV outbreaks (GII.4) were serially diluted and inoculated on the surface of lettuce pieces of a 9 cm² size. Inoculum concentration ranged from 1-5 log genome equivalent copies per sample. Viruses were pre-concentrated by sequential elution using 25 ml of 0.5 M glycine-0.14 M NaCl  buffer (pH 9.0) and precipitation with 12% polyethylene glycol.  After exposure to 5’ biotinylated aptamer candidates AP 25 or M6-2, the virus-aptamer conjugate was captured using streptavidin coated magnetic beads. Viral RNA was extracted using the NucliSENS^®easyMAG system and detected by RT-qPCR targeting the virus ORF1/ORF2 junction.

Results: The percent capture efficiency for AMC ranged from 2.5-35% for aptamer AP25 and between 1.5-7.0% for aptamer M2-6.  Capture efficiency improved with decreasing virus concentration.  Detection limits for the combined AMC-RT-qPCR assay were 1-2 log genome equivalent copies per lettuce sample. 

Significance: Both aptamer candidates show promise for the concentration and purification of low concentrations of HuNoV from foods prior to detection using RT-qPCR.