Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

Abstract

BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test.OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses.METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection.FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

Within-host genomics of SARS-CoV-2

“…Residual RNA from COVID-19 RT-qPCR-based testing was obtained from Oxford University Hospitals ('Oxford'), extracted on the QIASymphony platform with QIAsymphony DSP Virus/Pathogen Kit (QIAGEN), and from Basingstoke and North Hampshire Hospital ('Basingstoke'), extracted with one of: Maxwell RSC Viral total nucleic acid kit (Promega); Reliaprep blood gDNA miniprep system (Promega); or Prepito NA body fluid kit (PerkinElmer). An internal extraction control was added to the lysis buffer prior to extraction to act as a control for extraction efficiency (genesig qRT-PCR kit, #Z-Path-2019-nCoV in Basingstoke, MS2 bacteriophage ( 37 ) in Oxford). The #Z-Path-2019-nCoV control is a linear, synthetic RNA target based on sequence from the rat ptprn2 gene, which has no sequence similarity with SARS-CoV-2 (GENESIG PrimerDesign pers.…”

“…comm, 6 April 2020). The MS2 RNA likewise has no SARS-CoV-2 similarity ( 37 ) . Neither control RNA interfered with sequencing.…”

Development of a universal internal positive control

“…Therefore future developmental efforts might also include adapting the described IPC for use as a universal, nonhomologous exogenous internal control for RT-PCR. One suggested approach might involve RNA synthesis of the forward and/or reverse IPC template (Figure 1) with base modifications, such asphosphorothioate bonds [32], to convey resistance to degradation by pervasive RNases and ensure positivity of the control, similar in concept to IPC RNA templates constructed with a degradation protective protein shell [26,27,31]. The IPC RNA template would be spiked into a sample with subsequent RNA extraction and purification followed by a one- or two-step RT-PCR involving cDNA synthesis via random primers or the IPC primer(s) (Figure 1) and qPCR (Table 1).…”

External Control Viral-Like Particle Construction for Detection of Emergent Arboviruses by Real-Time Reverse-Transcription PCR

“…We proposed an external positive control based on the MS2 bacteriophage modified. Generally, this strategy has been chosen when only one target is being assayed, but we developed our control to cover all seven targets in 4 multiplex RT-qPCR assays, based on the first control of our group that was used in HCV RT-qPCR assay [12]. The stability of our control can be highlighted because until now it was maintained for 12 months.…”

“…The pET47b(+)-MS2-ZDC vector was transformed into NiCo21(DE3)-competent Escherichia coli in accordance with the manufacturer's instructions (New England Biolabs, USA). The protocol for expression and purification was described previously by Zambenedetti et al [12], with some modifications. The expression of pET47b(+)-MS2-ZDC was induced by the addition of 0.5 mM isopropyl-1- β -D-thiogalactoside (IPTG), and after centrifugation, the supernatant was collected and processed with filtration for viral-like particles purification.…”