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Identification of Clostridium beijerinckii, Cl. butyricum, Cl. sporogenes, Cl. tyrobutyricum isolated from silage, raw milk and hard cheese by a multiplex PCR assay

Published online by Cambridge University Press:  05 July 2012

Paola Cremonesi ,
Laura Vanoni ,
Tiziana Silvetti ,
Stefano Morandi  and
Milena Brasca
Paola Cremonesi
Affiliation:
Institute of Agricultural Biology and Biotechnology–Italian National Research Council, Via Bassini 15, Milan, Italy
Laura Vanoni
Affiliation:
Institute of Sciences of Food Production–Italian National Research Council, Via Celoria 2, Milan, Italy
Tiziana Silvetti
Affiliation:
Institute of Sciences of Food Production–Italian National Research Council, Via Celoria 2, Milan, Italy
Stefano Morandi
Affiliation:
Institute of Sciences of Food Production–Italian National Research Council, Via Celoria 2, Milan, Italy
Milena Brasca*
Affiliation:
Institute of Sciences of Food Production–Italian National Research Council, Via Celoria 2, Milan, Italy
*
*For correspondence; e-mail: milena.brasca@ispa.cnr.it
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Abstract

Late blowing, caused by the outgrowth of clostridial spores present in raw milk and originating from silage, can create considerable product loss, especially in the production of hard and semi-hard cheeses. The conventional method for the isolation of Clostridium spp. from cheeses with late-blowing symptoms is very complicated and the identification of isolates is problematic. The aim of this work was the development of a multiplex PCR method for the detection of the main dairy-related clostridia such as: Cl. beijerinckii, Cl. butyricum, Cl. sporogenes, Cl. tyrobutyricum. Samples derived from silage, raw milk and hard cheese were analysed by the most probable number (MPN) enumeration. Forty-four bacterial strains isolated from gas positive tubes were used to check the reliability of the multiplex PCR assay. The specificity of the primers was tested by individually analysing each primer pair and the primer pair combined in the multiplex PCR. It was interesting to note that the samples not identified by the multiplex PCR assay were amplified by V2–V3 16S rRNA primer pair and the sequencing revealed the aligned 16S rRNA sequences to be Paenibacillus and Bacillus spp. This new molecular assay provides a simple promising alternative to traditional microbiological methods for a rapid, sensitive detection of clostridia in dairy products.

Keywords

Clostridium spp.multiplex PCRraw milksilagecheese
Type
Research Article
Information
Journal of Dairy Research , Volume 79 , Issue 3 , August 2012 , pp. 318 - 323
Copyright
Copyright © Proprietors of Journal of Dairy Research 2012

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