Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantification of Enteric RNA Viruses in Different Matrices

Abstract

The detection and quantification of enteric RNA viruses is based on isolation of viral RNA from the sample followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). To control the whole process of analysis and in order to guarantee the validity and reliability of results, process control viruses (PCV) are used. The present article describes the process of preparation and use of such PCV– MS2 phage-like particles (MS2 PLP) – in RT-qPCR detection and quantification of enteric RNA viruses. The MS2 PLP were derived from bacteriophage MS2 carrying a unique and specific de novo-constructed RNA target sequence originating from the DNA of two extinct species. The amount of prepared MS2 particles was quantified using four independent methods – UV spectrophotometry, fluorimetry, transmission electron microscopy and a specifically developed duplex RT-qPCR. To evaluate the usefulness of MS2 PLP in routine diagnostics different matrices known to harbor enteric RNA viruses (swab samples, liver tissue, serum, feces, and vegetables) were artificially contaminated with specific amounts of MS2 PLP. The extraction efficiencies were calculated for each individual matrix. The prepared particles fulfill all requirements for PCV – they are very stable, non-infectious, and are genetically distinct from the target RNA viruses. Due to these properties they represent a good morphological and physiochemical model. The use of MS2 PLP as a PCV in detection and quantification of enteric RNA viruses was evaluated in different types of matrices.

Foodborne Bacterial, Viral, and Protozoan Pathogens in Field and Market Strawberries and Environment of Strawberry Farms

“…Tris base‐glycine beef extract (80 mL; pH 9.5) and pectinase (1368 μl; Pectinex™ Ultra SPL solution, Sigma, St. Louis, MO, U.S.A.) were added to 100 g of strawberries. This process was preceded by artificial contamination of each sample with 5 μL of process control virus (PCV, 10 6 particles/μL) to verify the isolation of viral RNA (Mikel et al., ). Samples were shaken (Biosan, Riga, Latvia) for 20 minutes to ensure proper elution of microorganisms.…”

“…The absolute quantification of chosen bacteria and parasites was carried out based on a calibration curve derived from a 10‐fold diluted plasmid standard. Detection and quantification of PCV, and calculation of processing efficiency was performed as previously (Mikel et al., ). Samples with processing efficiency <1% were not considered for further analysis.…”

Virucidal Influence of Ionic Liquids on Phages P100 and MS2

“…It is known to be stable at high sodium chloride concentrations (up to saturated solutions; EFSA, 2012 ). Phage MS2, a common surrogate for enteric RNA viruses, especially human noroviruses (Bae and Schwab, 2008 ; Mikel et al, 2016 ), has an isometric shape, +ssRNA, a diameter of 26 nm (Dawson et al, 2005 ) and is stable in monovalent salt concentrations of at least 1 M (Mylon et al, 2010 ). The aim of the study was to investigate if known IL structure activity relationship (SAR) outcomes can be applied to viruses or if additional or new SAR can be found.…”

Monitoring of selected parasites and other foodborne microorganisms in vegetable products at point of sale

“…One hundred grams of each sample were placed in a stomacher bag and artificially contaminated with 5 µL of process control virus (PCV, 106 particles/µL) to verify analysis for the presence of viral pathogens [29]. Subsequently, 230 mL of Trisma base-glycine beef extract (TGBE washing buffer, pH 9.5, in house) was added and homogenized for 2 min in order to remove microorganisms from vegetable surfaces.…”

“…Quantification of PCV as well as calculation of processing efficiency (estimate of PCV recovery) was done as defined by Mikel et al [29]. The samples where processing efficiency was lower than 1% were not considered as valid results and not included in further analyses.…”