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Rapid and direct quantitative detection of viable bifidobacteria in probiotic yogurt by combination of ethidium monoazide and real-time PCR using a molecular beacon approach

Published online by Cambridge University Press:  08 September 2010

XC Meng ,
R Pang ,
C Wang  and
LQ Wang
XC Meng*
Affiliation:
Key Laboratory of Dairy Science (Ministry of Education), Northeast Agricultural University, Harbin, People's Republic of China
R Pang
Affiliation:
Key Laboratory of Dairy Science (Ministry of Education), Northeast Agricultural University, Harbin, People's Republic of China
C Wang
Affiliation:
Key Laboratory of Dairy Science (Ministry of Education), Northeast Agricultural University, Harbin, People's Republic of China
LQ Wang
Affiliation:
Key Laboratory of Dairy Science (Ministry of Education), Northeast Agricultural University, Harbin, People's Republic of China
*
*For correspondence; e-mail: [email protected]
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Abstract

The potential of ethidium monoazide (EMA) real-time PCR method based on molecular beacon probe for rapid detection of viable bifidobacteria present in probiotic yogurt was evaluated in this work. A real-time PCR with molecular beacon assay was developed to determine genus Bifidobacterium quantitatively in order to increase the sensitivity and specificity of assay. EMA was used to treat probiotic yogurt prior to DNA extraction and real-time PCR detection to allow detection of only viable bacteria. The primer set of Bif-F/Bif-R which is genus-specific for Bifid. was designed. The specificity of the probes ensures that no signal is generated by non-target amplicons. Linear regression analysis demonstrated a good correlation (R2=0·9948) between the EMA real-time PCR results and the plate counting, and real-time quantitative PCR results correlated adequately with enumeration of bifidobacteria by culture for commercial probiotic yogurt. This culture-independent approach is promising for the direct and rapid detection of viable bifidobacteria in commercial probiotic yogurt, and the detection can be carried out within 4 h. The detection limit for this method is about 104 cell/ml. In conclusion, the direct quantitative EMA real-time PCR assay based on molecular beacon described in this research is a rapid and quantitative method.

Keywords

Bifidobacteriumreal-time PCRmolecular beaconethidium monoazide
Type
Research Article
Information
Journal of Dairy Research , Volume 77 , Issue 4 , November 2010 , pp. 498 - 504
Copyright
Copyright © Proprietors of Journal of Dairy Research 2010

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