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A Membrane-Based Electro-Separation Method (MBES) for Sample Clean-Up and Norovirus Concentration.

Kang W, Cannon JL - PLoS ONE (2015)

Bottom Line: As high as 30.8% MNV-1 migrated from the 3.5 ml sample chamber to the 1.5 ml collection chamber across a 1 μm separation membrane when 20 V was applied for 30 min using 20 mM sodium phosphate with 0.01% SDS (pH 7.5) as the electrolyte.The electric field strength of the system was the key factor to enhance virus movement, which could only be improved by shortening the electrodes distance, instead of increasing system applied voltage because of virus stability.This study successfully demonstrated the norovirus mobility in an electric field and migration across a size-specific membrane barrier in sodium phosphate electrolyte.

View Article: PubMed Central - PubMed

Affiliation: Center for Food Safety, The University of Georgia, Griffin, Georgia, United States of America.

ABSTRACT
Noroviruses are the leading cause of acute gastroenteritis and foodborne illnesses in the United States. Enhanced methods for detecting noroviruses in food matrices are needed as current methods are complex, labor intensive and insensitive, often resulting in inhibition of downstream molecular detection and inefficient recovery. Membrane-based electro-separation (MBES) is a technique to exchange charged particles through a size-specific dialysis membrane from one solution to another using electric current as the driving force. Norovirus has a net negative surface charge in a neutrally buffered environment, so when placed in an electric field, it moves towards the anode. It can then be separated from the cathodic compartment where the sample is placed and then collected in the anodic compartment for downstream detection. In this study, a MBES-based system was designed, developed and evaluated for concentrating and recovering murine norovirus (MNV-1) from phosphate buffer. As high as 30.8% MNV-1 migrated from the 3.5 ml sample chamber to the 1.5 ml collection chamber across a 1 μm separation membrane when 20 V was applied for 30 min using 20 mM sodium phosphate with 0.01% SDS (pH 7.5) as the electrolyte. In optimization of the method, weak applied voltage (20 V), moderate duration (30 min), and low ionic strength electrolytes with SDS addition were needed to increase virus movement efficacy. The electric field strength of the system was the key factor to enhance virus movement, which could only be improved by shortening the electrodes distance, instead of increasing system applied voltage because of virus stability. This study successfully demonstrated the norovirus mobility in an electric field and migration across a size-specific membrane barrier in sodium phosphate electrolyte. With further modification and validation in food matrixes, a novel, quick, and cost-effective sample clean-up technique might be developed to separate norovirus particles from food matrices by electric force.

No MeSH data available.


Related in: MedlinePlus

Devices used to investigate viral particles movement in electric field without the presence of membrane barrier.(A) illustration of the U-shape device; (B) illustration of the horizontal device; (C) actual picture of the U-shape device; (D) actual picture of the horizontal device.
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pone.0141484.g003: Devices used to investigate viral particles movement in electric field without the presence of membrane barrier.(A) illustration of the U-shape device; (B) illustration of the horizontal device; (C) actual picture of the U-shape device; (D) actual picture of the horizontal device.

Mentions: In a subset of experiments, a simple device was constructed to evaluate norovirus mobility in electric field without the effect of physical membrane barrier. Two 5 ml syringe barrels (Becton, Dickinson and Company, Franklin Lakes, NJ) were connected with a 6 cm tubing (1/2” ID × 5/8” OD × 1/16” wall Tygon®S3TM E-3603 laboratory tubing; United States Plastic Corp, Lima, OH). Platinum electrode wires connected to the power supply were inserted into the open ends of the syringe barrels to create an electric circuit. In one set of experiments, the device was bent into a U-shape and fixed on a rack (Fig 3A and 3C). Sodium phosphate buffer (500 mM, pH 7.5) with 0.01% SDS was filled into the device followed by 50 μl (7 log genomic copies) of MNV-1 addition to the syringe barrel with negative electrode wire (cathode) inserted. 20V constant voltage (corresponding to 18 mA electric current) was applied to the U-shape device for 30 min. Then the middle section of the tubing was clamped by an office clamp to stop the fluidic flow. After removing the applied electricity, the total sample volumes from the cathode and anode compartments were collected for RNA extraction and RT-qPCR detection. In the second set of experiments, the U-shape device was modified and a horizontal device was constructed. The plungers for the two syringe barrels were inserted to the 6 cm tubing (described above) to prevent liquid flow out of the tubing. Platinum wire, connected to a power supply, was inserted into holes drilled on each plunger (self-sealed with its rubber gasket) to establish an electric circuit (Fig 3B and 3D). Holes were punched in the top of the horizontally placed tubing to allow the passage of gas generated when the electric current was applied. Sodium phosphate buffer with 0.01% SDS (60 mM, pH 7.5) was filled into the tubing along with 50 μl (7 log genomic copies) MNV-1. 40V (corresponding to 30 mA electric current) was applied to the system for 10 min and 30 min, then the middle section of the tubing was clamped and the samples processed as described above. In both sets of experiments, no voltage (0V) was applied for 30 min to serve as the control for assessing virus movement by simple diffusion in the system.


A Membrane-Based Electro-Separation Method (MBES) for Sample Clean-Up and Norovirus Concentration.

Kang W, Cannon JL - PLoS ONE (2015)

Devices used to investigate viral particles movement in electric field without the presence of membrane barrier.(A) illustration of the U-shape device; (B) illustration of the horizontal device; (C) actual picture of the U-shape device; (D) actual picture of the horizontal device.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4625962&req=5

pone.0141484.g003: Devices used to investigate viral particles movement in electric field without the presence of membrane barrier.(A) illustration of the U-shape device; (B) illustration of the horizontal device; (C) actual picture of the U-shape device; (D) actual picture of the horizontal device.
Mentions: In a subset of experiments, a simple device was constructed to evaluate norovirus mobility in electric field without the effect of physical membrane barrier. Two 5 ml syringe barrels (Becton, Dickinson and Company, Franklin Lakes, NJ) were connected with a 6 cm tubing (1/2” ID × 5/8” OD × 1/16” wall Tygon®S3TM E-3603 laboratory tubing; United States Plastic Corp, Lima, OH). Platinum electrode wires connected to the power supply were inserted into the open ends of the syringe barrels to create an electric circuit. In one set of experiments, the device was bent into a U-shape and fixed on a rack (Fig 3A and 3C). Sodium phosphate buffer (500 mM, pH 7.5) with 0.01% SDS was filled into the device followed by 50 μl (7 log genomic copies) of MNV-1 addition to the syringe barrel with negative electrode wire (cathode) inserted. 20V constant voltage (corresponding to 18 mA electric current) was applied to the U-shape device for 30 min. Then the middle section of the tubing was clamped by an office clamp to stop the fluidic flow. After removing the applied electricity, the total sample volumes from the cathode and anode compartments were collected for RNA extraction and RT-qPCR detection. In the second set of experiments, the U-shape device was modified and a horizontal device was constructed. The plungers for the two syringe barrels were inserted to the 6 cm tubing (described above) to prevent liquid flow out of the tubing. Platinum wire, connected to a power supply, was inserted into holes drilled on each plunger (self-sealed with its rubber gasket) to establish an electric circuit (Fig 3B and 3D). Holes were punched in the top of the horizontally placed tubing to allow the passage of gas generated when the electric current was applied. Sodium phosphate buffer with 0.01% SDS (60 mM, pH 7.5) was filled into the tubing along with 50 μl (7 log genomic copies) MNV-1. 40V (corresponding to 30 mA electric current) was applied to the system for 10 min and 30 min, then the middle section of the tubing was clamped and the samples processed as described above. In both sets of experiments, no voltage (0V) was applied for 30 min to serve as the control for assessing virus movement by simple diffusion in the system.

Bottom Line: As high as 30.8% MNV-1 migrated from the 3.5 ml sample chamber to the 1.5 ml collection chamber across a 1 μm separation membrane when 20 V was applied for 30 min using 20 mM sodium phosphate with 0.01% SDS (pH 7.5) as the electrolyte.The electric field strength of the system was the key factor to enhance virus movement, which could only be improved by shortening the electrodes distance, instead of increasing system applied voltage because of virus stability.This study successfully demonstrated the norovirus mobility in an electric field and migration across a size-specific membrane barrier in sodium phosphate electrolyte.

View Article: PubMed Central - PubMed

Affiliation: Center for Food Safety, The University of Georgia, Griffin, Georgia, United States of America.

ABSTRACT
Noroviruses are the leading cause of acute gastroenteritis and foodborne illnesses in the United States. Enhanced methods for detecting noroviruses in food matrices are needed as current methods are complex, labor intensive and insensitive, often resulting in inhibition of downstream molecular detection and inefficient recovery. Membrane-based electro-separation (MBES) is a technique to exchange charged particles through a size-specific dialysis membrane from one solution to another using electric current as the driving force. Norovirus has a net negative surface charge in a neutrally buffered environment, so when placed in an electric field, it moves towards the anode. It can then be separated from the cathodic compartment where the sample is placed and then collected in the anodic compartment for downstream detection. In this study, a MBES-based system was designed, developed and evaluated for concentrating and recovering murine norovirus (MNV-1) from phosphate buffer. As high as 30.8% MNV-1 migrated from the 3.5 ml sample chamber to the 1.5 ml collection chamber across a 1 μm separation membrane when 20 V was applied for 30 min using 20 mM sodium phosphate with 0.01% SDS (pH 7.5) as the electrolyte. In optimization of the method, weak applied voltage (20 V), moderate duration (30 min), and low ionic strength electrolytes with SDS addition were needed to increase virus movement efficacy. The electric field strength of the system was the key factor to enhance virus movement, which could only be improved by shortening the electrodes distance, instead of increasing system applied voltage because of virus stability. This study successfully demonstrated the norovirus mobility in an electric field and migration across a size-specific membrane barrier in sodium phosphate electrolyte. With further modification and validation in food matrixes, a novel, quick, and cost-effective sample clean-up technique might be developed to separate norovirus particles from food matrices by electric force.

No MeSH data available.


Related in: MedlinePlus