Real-time PCR provides improved detection and titer determination of bacteriophage.
The plaque assay is the traditional method for the quantification of bacteriophage, particularly for lambda cloning vectors. Unfortunately, this technique is fraught with procedural difficulties, and the quality of the data obtained from this "gold standard" assay may be inaccurate due to the subjective interpretation of the results. The application of quantitative real-time PCR (QPCR) technology can address these issues and be a more accurate platform to evaluate phage growth conditions and quantify viral titers in phage preparations. QPCR, with an improved primer set specific for lambda phage and coupled with fluorescent dye detection of PCR products, was used to detect and quantify phages in lysates with no prior DNA purification. Phages were detected below one plaque-forming unit, and at least 89 viral copies were detected from a purified DNA sample. When unknown concentrations of various phage preparations were assessed using QPCR, they were attained more efficiently, with greater sensitivity and precision, and the method produced more accurate quantitative data spanning a wider linear range than those obtained by the plaque assay (six logs vs. one log, respectively). Finally, QPCR for the detection of phage has multiple applications, including conventional cloning and in alternative fields of study such as environmental sciences.
Edelman
Daniel C
DC
Department of Pathology, School of Medicine, University of Maryland Baltimore, 725 West Lombard Street, Rm S407, Baltimore, MD 21201, USA. dedelman@umaryland.edu
Barletta
Janet
J
D010582:Bacteriophage lambda / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D002498:Centrifugation; D004279:DNA, Viral / Q000302:isolation & purification; D003850:Deoxyribonuclease I / Q000378:metabolism; D005456:Fluorescent Dyes; D019288:Herpesvirus 8, Human / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D010948:Viral Plaque Assay
D004279:DNA, Viral; D005456:Fluorescent Dyes; D003850:Deoxyribonuclease I