Enumeration of bacteriophage particles: Comparative analysis of the traditional plaque assay and real-time QPCR- and nanosight-based assays

Abstract

Bacteriophages are increasingly being utilized and considered for various practical applications, ranging from decontaminating foods and inanimate surfaces to human therapy; therefore, it is important to determine their concentrations quickly and reliably. Traditional plaque assay (PA) is the current "gold standard" for quantitating phage titers. However, it requires at least 18 h before results are obtained, and they may be significantly influenced by various factors. Therefore, two alternative assays based on the quantitative real-time polymerase chain reaction (QPCR) and NanoSight Limited (NS) technologies were recently proposed for enumerating phage particles. The present study compared the three approaches' abilities to quantitate Listeria monocytogenes-, Escherichia coli O157:H7- and Yersinia pestis-specific lytic phages quickly and reproducibly. The average coefficient of variation (CVS) of the PA method including all three phages was 0.15. The reproducibility of the PA method decreased dramatically when multiple investigators performed the assays, and mean differences of as much as 0.33 log were observed. The QPC R method required costly equipment and the synthesis of phage-specific oligonucleotide primers, but it determined phage concentrations faster (within about 4 h) and more precisely than did PA (CVS = 0.13). NS technology required costly equipment, was less precise (CVS = 0.28) than the PA and QPCR methods, and only worked when the phages were suspended in clear medium. However, it provided results within 5 min. After the overall correlation is established with the PA method, either of the two assays may be useful for quickly and reproducibly determining phage concentrations.

Figure 1

Range of possible variance in phage titers determined by the PA. The diagonal line represents perfect agreement. The numeric values before and after the semi-colons were obtained by two different investigators.

Figure 2

Correlation between the results obtained by the PA and by the NS- and QPCR-based assays for determining phage titers. The symbols represent matched-pair, ten-point data set means plotted on a log scale. The diagonal lines represent perfect agreement. (A) Results obtained with the NS method and the PA. (B) Results obtained with the QPC R method and the PA. (C) Results obtained with the NS- and QPC R-based assays.