Optimization of an Adsorption–Elution Method with a Negatively Charged Membrane to Recover Norovirus from Lettuce

Abstract

Viral pathogens, such as norovirus (NoV), are frequently associated with foodborne gastroenteritis worldwide, and the detection of NoV in food requires appropriate methods and the use of process controls. In this study, an adsorption-elution concentration method using negatively charged membranes was optimized to recover NoV from lettuce, using murine norovirus 1 (MNV-1) as a human NoV (HuNoV) surrogate. Initially, three elution buffers were evaluated by direct elution using a Stomacher apparatus with a filter bag and different concentrations of MNV-1 genomic copies. The eluates were filtered in a Stericup and concentrated by a Centriprep Concentrator, and the viral RNA was quantified by real-time PCR that was preceded by reverse transcription. The MNV-1 recovery efficiency varied based on the buffers used, ranging from 5.2 to 9.8 % for PBS pH 7.2, 0.2-18 % for glycine NaCl pH 9.5 and 10.8-33.3 % for glycine Tris-HCl pH 9.5. Further analysis of the glycine Tris-HCl pH 9.5 buffer revealed that gentle-shaking, direct elution could replace the use of a Stomacher, with recovery rates reaching 66 and 32 % for MNV-1 and HuNoV, respectively, all of which suggested that this procedure is a quick and efficient method for recovering NoV from lettuce.

Evaluation of skimmed milk flocculation method for virus recovery from tomatoes

“…MNV-1 has been used as SPCV in other matrices, showing a good recovery percentage ranging from 7.78% to 75.65% 35 and 8.4% to 66.4%. 24 …”

“…MNV-1 experiments were also performed to evaluate success rate and recovery efficiency using methodology described by ISO 15216:2017 with results lower than those obtained by the organic flocculation method (data not shown). MNV-1 has been used as SPCV in other matrices, showing a good recovery percentage ranging from 7.78% to 75.65% 35 and 8.4% to 66.4% 24 …”

“…A NoV GII.4 stool sample (GenBank accession number JX975591) was obtained from the Regional Reference Gastroenteritis Laboratory collection, at Oswaldo Cruz Institute, Rio de Janeiro-RJ, Brazil. Murine norovirus-1 (MNV-1) was kindly provided by Dr. Herbert W. Virgin (Washington University School of Medicine) and propagated in RAW 264.7 cells (a macrophage-like Abelson leukemia virus-transformed cell line derived from BALB/c mice; ATCC ® TIB-71™), according to de Abreu Corrêa and Miagostovich 24 . HAdVs type 2 was propagated in HEK 293 cells (human embryonic kidney cells; ATCC ® CRL1573™) obtained from the Regional Reference Gastroenteritis Laboratory collection, at Oswaldo Cruz Institute, Rio de Janeiro, RJ, Brazil 30 …”

“…The increasing number of viruses foodborne outbreaks have resulted in a growing number of studies that evaluate elution and concentration methods from different food matrices, as well as the use of sample processes control viruses (SPCVs) as murine norovirus-1 (MNV-1), bacteriophage PP7 and others. 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 In 2013, the International Organization for Standardization (ISO), together with the European Committee for Standardization (CEN), standardized methodologies for recovering NoV and hepatitis A virus from matrix foods 27 that were validated recently. 28…”

Adsorption and Elution of Nucleic Acids: Mesoporous Materials and Methods

“…It has been argued that nucleic acid interactions with silicate materials are via amine and carboxyl groups [68]. Methods used to displace these interactions as well as those used to displace RNA from negatively charged membranes [69] were considered and tested without improvement. Finally, various nucleic acid washing solutions and hybridization buffers were evaluated without success (less than 1% of target recovered).…”