A qPCR Targeted Against the Viral Replication Origin Designed to Quantify Total Amount of Filamentous Phages and Phagemids.

Abstract

Filamentous bacteriophages are widely used in phage display technology. The most common quantification method is lysis plaque formation test (PFT). This technique has several disadvantages, and only quantifies infective phages and is not effective when phagemids are used. We developed a qPCR method directed against the M13 replication origin, which detects between 3.3 × 103 and 3.3 × 108 viral genome copies with a linearity of R 2 = 0.9998. Using this method we were able to observe a difference of approximately ten more phages than with the PFT. This difference was not due to the presence of a free genome, which suggests the presence of non-infective particles. Using a DNaseI treatment, we observed the presence of 30% to 40% of unpackaged genome in recombinant phage modified in PIII or PVIII. The qPCR method with a DNase I treatment is an efficient method to quantify the total amount of filamentous phages.