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

J E Méndez-Scolari1, M M Florentín-Pavía1, M P Mujica1

  • 1Dpto. de Biotecnología, Facultad de Ciencias Químicas, Universidad Nacional de Asunción, Campus Universitario, San Lorenzo, Paraguay.

Indian Journal of Microbiology
|August 8, 2019
PubMed

Insights

A new qPCR method accurately quantifies filamentous phages, including non-infective particles. This technique improves phage display research by detecting more phages than traditional plaque formation tests.

Area of Science:

  • Molecular Biology
  • Biotechnology

Background:

  • Filamentous bacteriophages are crucial for phage display.
  • Plaque formation test (PFT) is the standard but has limitations.

Purpose of the Study:

  • Develop a more effective method for filamentous phage quantification.
  • Address limitations of PFT, especially for phagemids and non-infective particles.

Main Methods:

  • Developed a qPCR assay targeting the M13 replication origin.
  • Validated the qPCR method's sensitivity and linearity.
  • Employed DNase I treatment to assess unpackaged genomes.

Main Results:

  • qPCR detected 3.3x10^3 to 3.3x10^8 viral genome copies with high linearity (R²=0.9998).
  • qPCR identified approximately 10x more phages than PFT.
  • DNase I treatment revealed 30-40% unpackaged genomes in modified phages.

Conclusions:

  • qPCR offers a more sensitive and comprehensive quantification of filamentous phages.
  • The method detects both infective and non-infective phage particles.
  • Combined qPCR with DNase I treatment provides efficient total phage quantification.
Keywords:
Phage displayPhage quantitationPhagemidsqPCR

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