Multiplex PCR for detection and identification of lactococcal bacteriophages

Area of Science:

• Microbiology
• Molecular Biology
• Food Science

Background:

• Lactococcus lactis phages, specifically the 936, c2, and P335 species, are prevalent in dairy processing plants.
• Accurate and rapid detection methods are crucial for managing phage contamination in dairy production.

Purpose of the Study:

• To develop and validate a multiplex PCR assay for the simultaneous detection of the three main Lactococcus lactis phage species.
• To assess the efficiency and sensitivity of the assay in various dairy matrices.

Main Methods:

• Design of species-specific primers targeting conserved genomic regions (major capsid protein gene for c2, major structural protein gene for 936).
• Adaptation of a multiplex PCR protocol for detecting phages in whey samples and phage lysates.
• Sequencing and comparative analysis of target genes from isolated phages to confirm primer specificity and assess genomic diversity.

Main Results:

• The multiplex PCR method successfully detected the 936, c2, and P335 phage species in a single reaction.
• Detection limits in whey ranged from 10^4 to 10^7 PFU/ml, improving with a concentration step.
• High sequence identity was observed within target genes for c2 (86.4%) and 936 (82.2%) phages, while P335 phages showed genomic diversity but a conserved region was identified.

Conclusions:

• The developed multiplex PCR is a sensitive and specific tool for rapid phage detection in dairy environments.
• The method aids in monitoring and controlling phage contamination, contributing to dairy product quality and safety.
• Further investigation into P335 phage diversity may be warranted, but a conserved region allows for detection.