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A qRT‐PCR‐based method for the measurement of rrn operon copy number
Author(s) -
Zhang T.,
Shao M.F.,
Fang H.H.P.
Publication year - 2009
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2009.02613.x
Subject(s) - biology , polymerase chain reaction , real time polymerase chain reaction , escherichia coli , low copy number , microbiology and biotechnology , genome , gene , genetics
Aim: To develop a convenient and accurate method for estimating the rrn operon copy number ( Y rrn ) in cells of pure prokaryotic cultures based on quantitative real‐time polymerase chain reaction (qRT‐PCR). Methods & Results: Using Escherichia coli, the Y rrn of which is known to be 7, as a reference, the rrn concentrations of target species and E. coli in sample solutions were measured based on their respective threshold cycle numbers ( C t ), whereas the cell concentrations of both species were measured by microscopic counting after staining. The Y rrn of the target species was then calculated from the initial cell concentrations and the rrn concentrations of the target species and E. coli . Using this method, the Y rrn values of four species, i.e. Xanthomonas campestris , Staphylococcus aureus , Aeromonas hydrophila and Pseudomonas fluorescens , were estimated as 1·80, 4·73, 8·58 and 5·13, respectively, comparable to their respective known values of 2, 5, 10, and 5, resulting in an average deviation of 8%. Conclusions: The whole cell qRT‐PCR based methods were convenient, accurate and reproducible in quantification of rrn copy number of prokaryotic cells. Significance and Impact of the Study: qTR‐PCR is a fast and reliable DNA quantification approach. Compared with previous qTR‐PCR based methods measuring rrn copy number, the present method avoided the prerequisite for the information on genome size and GC content of target bacteria or a gene with known copy number, thus should be more widely applicable.