Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays

Abstract

Diagnostic systems based on reverse transcription (RT)-PCR are widely used for the detection of viral genomes in different human specimens. The application of internal controls (IC) to monitor each step of nucleic acid amplification is necessary to prevent false-negative results due to inhibition or human error. In this study, we designed various real-time RT-PCRs utilizing the coliphage MS2 replicase gene, which differ in detection format, amplicon size, and efficiency of amplification. These noncompetitive IC assays, using TaqMan, hybridization probe, or duplex scorpion probe techniques, were tested on the LightCycler and Rotorgene systems. In our approach, clinical specimens were spiked with the control virus to monitor the efficiency of extraction, reverse transcription, and amplification steps. The MS2 RT-PCR assays were applied for internal control when using a second target hepatitis C virus RNA in duplex PCR in blood donor screening. The 95% detection limit was calculated by probit analysis to 44.9 copies per PCR (range, 38.4 to 73.4). As demonstrated routinely, application of MS2 IC assays exhibits low variability and can be applied in various RT-PCR assays. MS2 phage lysates were obtained under standard laboratory conditions. The quantification of phage and template RNA was performed by plating assays to determine PFU or via real-time RT-PCR. High stability of the MS2 phage preparations stored at ؊20°C, 4°C, and room temperature was demonstrated.

High-Volume Extraction of Nucleic Acids by Magnetic Bead Technology for Ultrasensitive Detection of Bacteria in Blood Components

“…In gene expression analysis and virus screening, so-called housekeeping genes such as ␤-actin or glyceraldehyde 3-phosphate dehydrogenase are often used as ICs. This approach is suitable for specimens containing large amounts of human nucleic acids, such as PCs (31 ).…”

Evaluation of Human Enteric Viruses in Surface Water and Drinking Water Resources in Southern Ghana

“…The use of an RNA or DNA internal standard during PCR and RT-PCR has been a common method reported for evaluating sample inhibition in complex environmental and clinical matrices. 32,[49][50][51] However, this step is often missing during evaluation of environmental water samples. Identification of inhibition is imperative when reporting results that could potentially impact public health, especially if false negatives are reported.…”

Detection of polyoma and corona viruses in bats of Canada

“…PCR products of the expected size were submitted to the NML genomics core facility and sequences were analysed by BLAST to determine if they resembled known viral sequences. Bacteriophage MS2 RNA, seeded into raw samples as a control for RNA extraction, was detected using quantitative RT-PCR (Dreier et al, 2005) by the virology facility at the NML.…”