Methods for Preparation of MS2 Phage-Like Particles and Their Utilization as Process Control Viruses in RT-PCR and qRT-PCR Detection of RNA Viruses From Food Matrices and Clinical Specimens

Abstract

RNA viruses are pathogenic agents of many serious infectious diseases affecting humans and animals. The detection of pathogenic RNA viruses is based on modern molecular methods, of which the most widely used methods are the reverse transcription polymerase chain reaction (RT-PCR) and the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). All steps of RT-PCR and qRT-PCR should be strictly controlled to ensure the validity of obtained results. False-negative results may be caused not only by inhibition of RT or/and PCR steps but also by failure of the nucleic acid extraction step, particularly in the case of viral RNA extraction. The control of nucleic acid extraction generally involves the utilization of a non-pathogenic virus (process control virus) of similar structural properties to those of the target virus. Although in clinical samples the use of such process control virus is only recommended, in other kinds of settings such as food matrices its use is necessary. Currently, several different process control viruses are used for these purposes. Process control viruses can also be constructed artificially using technology for production of MS2 phage-like particles, which have many advantages in comparison with other used controls and are especially suited for controlling the detection and quantification of certain types of RNA viruses. The technology for production of MS2 phage-like particles is theoretically well established, uses the knowledge gained from the study of the familiar bacteriophage MS2 and utilizes many different approaches for the construction of the various process control viruses. Nevertheless, the practical use of MS2 phage-like particles in routine diagnostics is relatively uncommon. The current situation with regard to the use of MS2 phage-like particles as process control viruses in detection of RNA viruses and different methods of their construction, purification and use are summarized and discussed in this review.

One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles

“…Currently, the production of MS2 PLP is based on various in vivo plasmid-driven packaging systems, which exploit the spontaneous assembly of MS2 coat protein dimers in the presence of the pac site inside Escherichia coli ( E . coli ) 10 . However, while each of these systems have their advantages, they are also characterized by various drawbacks, e.g., limited capacity for packaging of control ssRNA, lower efficiency of packaging, complicated system construction, or complicated and time-consuming purification of produced MS2 PLP.…”

“…However, this system still does not address a major drawback of current MS2 PLP preparations: the purification procedure. Therefore, produced MS2 PLP must be purified in a time-consuming and laborious ultracentrifugation step, which does not isolate MS2 PLP in the required purity 4 , 8 , 10 , 11 , 13 . The problem of purification can be solved by the introduction of an affinity tag (e.g., polyhistidine-tag; His-tag) to the structure of MS2 PLP allowing their purification using the Co 2+ affinity chromatography method 13 , 14 .…”

The True Story and Advantages of RNA Phage Capsids as Nanotools

“…Armored RNAs have multiple uses, such as for the detection of HCV, severe acute respiratory syndrome coronavirus, and influenza virus RNA [276] or for the creation of unique RNA molecules harboring both tRNA and mRNA functions [277]. Perspectives on the production and practical use of MS2 VLPs for routine diagnostics including food quality control were discussed in a recent review [278]. …”

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

“…The particles have similar physiochemical properties as the wild-type bacteriophage MS2, which has been used many times as a PCV in the detection of enteric RNA viruses (Dreier et al, 2006; Rolfe et al, 2007; Blaise-Boisseau et al, 2010; Shulman et al, 2012) and as the surrogate virus in enteric RNA virus environmental stability studies (Shin and Sobsey, 2003; Bae and Schwab, 2008; Park and Sobsey, 2011). The present article is a follow-up study of a previously published theoretical concept (Mikel et al, 2015) and describes a method of preparation of MS2 PLP carrying a specific control sequence and their use as a PCV in RT-qPCR detection and quantification of enteric RNA viruses in swab, liver tissue, serum, feces, and vegetable samples.…”