Development of quantitative PCR and metagenomics-based approaches for strain quantification of a defined mixed-strain starter culture

Abstract

Although the strain composition of mixed cultures may hugely affect production of various fermented foods, such as e.g. cheese, tools for investigating it have so far been limited. In this study, two new approaches for quantification of seven Lactococcus lactis subsp. cremoris strains (S1-S7) in a defined mixed-strain starter culture were developed and verified. By mapping NGS reads from 47 sequenced L. lactis strains to de novo assembly contigs of the seven strains, two strain-specific sequence regions (SEQ1 and SEQ2) were identified for each strain for qPCR primer design (A1 and A2). The qPCR assays amplified their strain-specific sequence region target efficiently. Additionally, high reproducibility was obtained in a validation sample containing equal amounts of the seven strains, and assay-to-assay coefficients of variance (CVs) for six (i.e. S1, S2, S4-S7) of the seven strains correlated to the inter-plate CVs. Hence, at least for six strains, the qPCR assay design approach was successful. The metagenomics-based approach quantified the seven strains based on average coverage of SEQ1 and SEQ2 by mapping sequencing reads from the validation sample to the strain-specific sequence regions. Average coverages of the SEQ1 and SEQ2 in the metagenomics data showed CVs of ≤17.3% for six strains (i.e. S1-S4, S6, S7). Thus, the metagenomics-based quantification approach was considered successful for six strains, regardless of the strain-specific sequence region used. When comparing qPCR- and metagenomics-based quantifications of the validation sample, the identified strain-specific sequence regions were considered suitable and applicable for quantification at a strain level of defined mixed-strain starter cultures.

Keywords: Defined mixed-strain starter culture; Genome sequencing; Metagenomics; Quantitative-PCR (qPCR); Strain quantification.