There are two common ways of genome characterization for bacteriophages: restriction analysis and complete genome sequencing. Does anyone know of an example of successful PCR diagnostics of bacteriophages with group-specific primers?
You would only need reverse transcriptase (RT in RT-PCR) if you wanted to detect messenger RNA transcripts of active phage. Most (or all?) phages are DNA, so you can use primers and PCR without RT, if you just want to detect phage in your sample. PCR can be very highly sensitive and able to pull out a single copy of the target DNA amid billion-fold excess of other DNA, but it is also highly prone to false positive results from contamination of the sample with trace amounts of target DNA. Even the very most careful labs have contamination events, so the trick is to devise methods (such as double blinding of samples/controls) to prove that contamination is not an issue.
Designing primers can be done with software to produce a multiple sequence alignment of your "group" and search for regions conserved (especially at the 3' end of each primer) across the entire group. See http://www.hiv.lanl.gov/tools/primer/main and
Identification of regions in multiple sequence alignments thermodynamically suitable for targeting by consensus oligonucleotides: application to HIV genome.
Matveeva OV, Foley BT, Nemtsov VA, Gesteland RF, Matsufuji S, Atkins JF, Ogurtsov AY, Shabalina SA.
BMC Bioinformatics. 2004 Apr 29;5:44.
PMID: 15115544
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