title: Development of a triplex real-time PCR assay for the simultaneous detection of Clostridium beijerinckii, Clostridium sporogenes and Clostridium tyrobutyricum in milk.
abstract: Clostridium beijerinckii, Clostridium sporogenes and Clostridium tyrobutyricum are considered the leading bacteria implicated in late blowing defects affecting semi-hard and hard cheese production. The aim of this study was to develop a multiplex Real-Time PCR (qPCR) analysis for a rapid and simultaneous detection of C. beijerinckii, C. sporogenes and C. tyrobutyricum, using specific primers respectively targeting the nifH, gerAA and enr genes. The limits of detection in raw milk were 300 CFU/50 mL in the case of C. beijerinckii, 2 CFU/50 mL for C. sporogenes and 5 CFU/50 mL for C. tyrobutyricum spores. The qPCR method was applied to artificially contaminated raw milk samples, and molecular quantification showed good correlation (R(2) = 0.978) with microbiological counting. Our results demonstrate that this method, combined with a DNA extraction protocol optimized for spore lysis, could be a useful tool for the direct quantification of the considered clostridia species.
authors: Institute of Sciences of Food Production, Italian National Research Council, Via Celoria 2, Milan, Italy.
authors: Stefano
authors: S
authors: Morandi
authors: Institute of Agricultural Biology and Biotechnology, Italian National Research Council, Via Bassini 15, Milan, Italy.
authors: Paola
authors: P
authors: Cremonesi
authors: Institute of Sciences of Food Production, Italian National Research Council, Via Celoria 2, Milan, Italy.
authors: Tiziana
authors: T
authors: Silvetti
authors: Institute of Agricultural Biology and Biotechnology, Italian National Research Council, Via Bassini 15, Milan, Italy.
authors: Bianca
authors: B
authors: Castiglioni
authors: Institute of Sciences of Food Production, Italian National Research Council, Via Celoria 2, Milan, Italy. Electronic address: milena.brasca@ispa.cnr.it.
authors: Milena
authors: M
authors: Brasca
mesh_terms: D000818:Animals; D058491:Bacterial Load; D003013:Clostridium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D005798:Genes, Bacterial; D008892:Milk / Q000382:microbiology*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers
keywords: Clostridium beijerinckii; Clostridium sporogenes; Clostridium tyrobutyricum; Raw milk; TaqMan(®) real-time quantitative PCR; Triplex real-time PCR