A Novel Membrane-based Electro-separation Method for Sample Clean-up and Norovirus Concentration

Introduction: Norovirus elution and concentration from complex food matrices is difficult, often resulting in inefficient recovery and inhibition in downstream molecular detection. Membrane-based electro-separation is a technique to exchange charged particles through membrane from one solution to another using electric current as the driving force. Norovirus has a net negative surface charge in neutral buffer environment and when placed in an electric field, it can move towards the anode. 

Purpose: Membrane-based electro-separation method was designed, developed and evaluated for its efficacy in concentrating and recovering noroviruses from phosphate buffer in a proof-of-concept study. 

Methods: ElectroPrep Electrodialysis system (Harvard Apparatus) was fitted with a 1 µm or 10 µm polycarbonate membrane (for pore-size selection) and two flanking 300 kDa MWCO cellular acetate restricting membranes as to create two chambers (one for sample addition (3.5 ml); one for sample collection (1.5 ml)). 10⁶ genome copies per ml of murine norovirus (MNV-1) was seeded into sample chamber and the linked chambers were submerged in sodium phosphate buffer (20 mM, 50 mM, 100 mM, 150 mM) containing 0.01% SDS (pH 7.5) as the electrolyte. Constant electric potential 20 V was provided for 30 min (0 V applied for 30 min as control). 500 µl was collected from each chamber for RNA extraction and quantification by RT-qPCR. All the experiments were performed in duplication. 

Results: %Recovery of MNV-1 from the collection chamber were 14.2%, 15.0%, 8.8%, and 16.0% with a 1 µm separation membrane and 27.6%, 24.3%, 21.6%, and 22.6% with a 10 µm separation membrane when 20 mM, 50 mM, 100 mM, and 150 mM electrolyte concentration were used. In control where 0 V were applied, 1.2% and 3.3% recoveries were obtained with 1 µm and 10 µm separation membranes, respectively, in 20 mM electrolyte. 

Significance: Norovirus recovery in the collection chamber is higher when electric potential is applied. With further optimization, membrane-based electro-separation method may provide a quick, easy, and cost-effective sample clean-up technique for norovirus detection.