Rapid Detection of Norovirus from Fresh Lettuce Using Immunomagnetic Separation and a Quantum Dots Assay

Abstract

Current molecular methods that include PCR have been used to detect norovirus in many food samples. However, the protocols require removing PCR inhibitors and incorporate time-consuming concentration steps to separate virus from analyte for rapid and sensitive detection of norovirus. We developed an immunomagnetic separation (IMS) and a quantum dots (QDs) assay to detect norovirus eluted from fresh lettuce with Tris buffer containing 1% beef extract (pH 9.5). IMS facilitated viral precipitation with a 10-min incubation, whereas virus concentration using polyethylene glycol (PEG) requires more than 3 h and an additional high-speed centrifugation step to precipitate virus before reverse transcription PCR (RT-PCR) analysis. The fluorescence intensity of QDs was detected qualitatively on norovirus dilutions of 10(-1) to 10(-3) in a stool suspension (100 RT-PCR units/ml). The results suggest that a fluorescence assay based on IMS and QDs is valid for detecting norovirus qualitatively according to fluorescent signal intensity within the same virus detection limit produced by IMS-RT-PCR and PEG-RT-PCR.

Selection, Characterization and Application of Nucleic Acid Aptamers for the Capture and Detection of Human Norovirus Strains

“…Use of aptamers for pre-concentration of microbes has been reported recently by others [36]. Comparatively speaking, the detection limit and capture efficiency of this method, at 10 RNA copies and 36%, respectively, were comparable to those for HuNoV immunomagnetic separation-RT-qPCR assays applied to artificially contaminated fresh produce items [29] [30] and clinical specimens [28]. Our detection limits were also similar if not better than those for other capture approaches that use more non-specific ligands such as histo-blood group antigens [37] [38] [39] and porcine gastric mucin [40].…”

“…Antibodies, the most frequently used ligands for HuNoV capture and detection [28] [29] [30], tend to lack broad reactivity, meaning that subsequent assays developed with these antibodies lack analytical sensitivity [6]. Having an alternative ligand type showing broad reactivity to multiple HuNoV strains provides another tool upon which capture and detection assays may be based.…”

Use of IHF-QD Microscopic Analysis for the Detection of Food Allergenic Components: Peanuts and Wheat Protein

“…The use of such QDs is widespread in the life sciences, but several publications have reported the use of QDs in the food sciences, where they were mostly used to detect pathogenic bacteria, proteins, and rotaviruses. They were also used in applying polyclonal antibody-bound QDs and to intensify the signal of detecting allergens [23,[25][26][27][29][30][31][32].…”

Ensemble and single-molecule biophysical characterization of D17.4 DNA aptamer–IgE interactions

“…The D17. 4 aptamer has been used as a recognition element in biosensing for sensitive IgE detection [18][19][20][21][22][23].…”

“…Most prior work on aptamer/protein interactions has focused on characterizing binding affinity, identifying residues involved in aptamer recognition, and application of aptamers in therapeutics [4,25] and biosensing [18][19][20][21]; relatively little is known about the kinetics and energetics of aptamer/protein interactions. In this work we studied the dissociation kinetics, the effects of temperature and ionic strength, and the kinetic activation parameters of the D17.…”