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.
Highlights
The quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is nowadays considered as the gold standard method for detection and quantification of enteric RNA viruses such as hepatitis A virus (HAV), hepatitis E virus (HEV) or human noroviruses (NoV) (Mattison et al, 2009; Blaise-Boisseau et al, 2010; Di Pasquale et al, 2010; Vasickova et al, 2012; HennechartCollette et al, 2014)
Because of the drawbacks of RT-qPCR including the necessity of monitoring the efficiency of concentration and RNA extraction steps, the removal of reverse transcription (RT) and PCR inhibitors there is a need of developing an entire RT-qPCR assay with a system of controls
Many different viruses were used as process control virus (PCV) for the RT-qPCR detection of HAV and NoV from food matrices, e.g., Murine norovirus 1 (MNV-1) (MartinLatil et al, 2012; Coudray et al, 2013; Hennechart-Collette et al, 2014), Feline calicivirus (FCV) (Mattison et al, 2009; Di Pasquale et al, 2010), Mengovirus, San Miguel sea lion virus serogroup 17 (SMSV-17) (DePaola et al, 2010) or echo type 9 virus (Nishida et al, 2007)
Summary
The quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is nowadays considered as the gold standard method for detection and quantification of enteric RNA viruses such as hepatitis A virus (HAV), hepatitis E virus (HEV) or human noroviruses (NoV) (Mattison et al, 2009; Blaise-Boisseau et al, 2010; Di Pasquale et al, 2010; Vasickova et al, 2012; HennechartCollette et al, 2014). In 2013 the European Committee for Standardization (CEN) released ISO technical specifications (ISO/TS) ISO/TS 15216-1 and ISO/TS 15216-2 (The methods for determination of HAV and NoV in food using RT-qPCR), which require the use of PCV together with external control RNA (EAC) in RT-qPCR detection of these viruses in such complex matrices According to these technical specifications, a cultivable non-enveloped positivesense single stranded RNA (+ssRNA) virus shall be used as such a control. The PCV should be of a similar size to the target virus to provide a good morphological and physicochemical model, should be genetically distinct from the target virus to avoid cross-reactivity, and should not be naturally present in the analyzed sample
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