Immunomagnetic Separation Combined with Real-Time Reverse Transcriptase PCR Assays for Detection of Norovirus in Contaminated Food

Abstract

We developed an immunomagnetic separation (IMS) technique combined with real-time TaqMan reverse transcriptase PCR (RT-PCR), which allowed detection of norovirus at a level as low as 3 to 7 RT-PCR units from artificially contaminated strawberries. The inoculum recovery rate ranged from 14 to 30%. The data demonstrate that IMS combined with real-time RT-PCR will be useful as a rapid and sensitive method for detecting food-borne microbial contaminants.

Detection of Noroviruses in Ready-To-Eat Foods by Using Carbohydrate-Coated Magnetic Beads

“…The methods developed here achieved detection within the range of the infectious dose for NoV and similar to the best methods for NoV detection from foods (15,19). This was demonstrated for multiple strains and food types (lettuce, green onion, and strawberries).…”

“…Methods used previously to concentrate NoV include ultracentrifugation (20), polyethylene glycol precipitation (8, 13), adsorption/elution (6), and immunomagnetic separation (14,19). These methods can be time-consuming (polyethylene glycol precipitation and adsorption/elution) and strain specific (immunomagnetic separation).…”

Enhancement of Enteric Adenovirus Cultivation by Viral Transactivator Proteins

“…ProSieve color protein markers (Cambrex, Walkersville, MD) were used as the molecular weight standards in 8% SDS-PAGE. Samples were subjected to SDS-PAGE on two gels for staining with Coomassie brilliant blue (Sigma Chemical Co., St. Louis, MO) and for Western blotting, as described previously (26). For immunoblotting, protein samples were transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham, Buckinghamshire, United Kingdom) using a Mini-PROTEAN 3 blot module (Bio-Rad Laboratories, Richmond, CA) and then blocked with 5% Difco skim milk (BD Biosciences, Mansfield, MA) in TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) for 1 h. Membranes were washed with TBS-0.1% Tween 20 (Sigma) three times for 10 min each time and then incubated for 1 h at room temperature with 6 ml of washing buffer and 4 ml of 5% skim milk containing a 1/1,000 dilution of anti-CMV IE monoclonal antibody (Chemicon, Temecula, CA).…”

Nucleic Acid Detection of Major Foodborne Viral Pathogens Human: Noroviruses and Hepatitis A Virus

“…There are two major groups of sequence-specific oligonucleotide probes: hydrolysis (e.g., TaqMan) probes and hybridization probes (e.g., molecular beacons and fluorescence resonance energy transfer probes); both groups are homologous to the internal region of amplified products. TaqMan probes have been frequently used in RT-qPCR for detecting hNOVs [67][68][69][70][71][72][73]. While RNA polymerase and capsid genes are the primary targets for amplification, within the NoV genomes, a junction of ORF1-ORF2-polymerase-capsid has been demonstrated to be the most highly conserved region that can serve as an effective target for amplification.…”