Quantification of Host and Phage mRNA Expression During Infection Using Real-Time PCR

Abstract

Real-time, or quantitative PCR is a valuable technique useful in bacteriophage research to quantify the abundance of phage or host gene transcripts. It can be used during the infection cycle both to monitor the expression of individual viral transcripts and to compare relative gene expression levels throughout the infection cycle. It is fairly economical to conduct and is useful in bacteria-phage systems where obtaining high yields of RNA is problematic. To perform real-time PCR it is simply necessary to know the DNA sequence of the genes to be monitored, to have accurately quantified mRNA and good quality cDNA, and access to a light-cycler. Although this chapter briefly reviews the basic principles of real-time PCR, the emphasis is on aspects of technique that are specific to the study of bacteriophage transcriptomics. These include (1) the 2 selection of the target gene, (2) the choice of calibrator and reference genes, and (3) RNA isolation for cDNA synthesis and (4) subsequent analysis of samples. This chapter should also be useful to those wishing to amplify genes from other types of templates such as metagenomic DNA or RNA extracted either from filtered samples or from agarose gels.

Biological Characterization and Evolution of Bacteriophage T7-△holin During the Serial Passage Process

“…The primer details are presented in Supplementary Table 1 . All data were analyzed statistically using the two-tailed Student’s t -test ( Clokie, 2009 ).…”

Real-Time qPCR as a Method for Detection of Antibody-Neutralized Phage Particles

“…This method can also be applied in other experiments that require detection of a “neutralized” phage, which is merely a phage that is not active against bacteria. Notably, qPCR has already been applied for phage detection in other (than phage neutralizing) conditions: in phage cultures ( Edelman and Barletta, 2003 ; Clokie, 2009 ; Anderson et al, 2011 ; Refardt, 2012 ; Dieterle et al, 2016 ), food ( Imamovic and Muniesa, 2011 ; Flannery et al, 2014 ; Perrin et al, 2015 ; Parente et al, 2016 ; Hartard et al, 2017 ; Muhammed et al, 2017 ), environmental samples ( Farkas et al, 2015 ; Kunze et al, 2015 ; Unnithan et al, 2015 ; Mankiewicz-Boczek et al, 2016 ), and in feces ( Imamovic et al, 2010 ; Chehoud et al, 2016 ), where some interference from antibodies cannot be excluded ( Majewska et al, 2015 ).…”

“…Real-time quantitative polymerase chain reaction (qPCR) detection of eukaryotic viruses in environmental and human or animal samples is a standard and commercialized method ( Watzinger et al, 2004 ; Hmaied et al, 2015 ). Bacteriophages have been quantitatively analyzed and discriminated by real-time qPCR directly in microbiological cultures, and the authors found this method to be a good alternative to the plaque assay ( Edelman and Barletta, 2003 ; Clokie, 2009 ; Anderson et al, 2011 ; Refardt, 2012 ; Dieterle et al, 2016 ). Real-time PCR has been further demonstrated as applicable for a rapid screening allowing phage detection in food (milk, fruits, vegetables, seafood, meat) ( Imamovic and Muniesa, 2011 ; Flannery et al, 2014 ; Perrin et al, 2015 ; Parente et al, 2016 ; Hartard et al, 2017 ) and water samples ( Farkas et al, 2015 ; Kunze et al, 2015 ; Unnithan et al, 2015 ; Mankiewicz-Boczek et al, 2016 ) or in feces ( Imamovic et al, 2010 ; Chehoud et al, 2016 ).…”