Comparative study of a new quantitative real-time PCR targeting the xylulose-5-phosphate/fructose-6-phosphate phosphoketolase bifidobacterial gene (xfp) in faecal samples with two fluorescence in situ hybridization methods

Valentine Cleusix 1 Christophe Lacroix 1, * G. Dasen 1 G. Leo 1 Gwenaëlle Le Blay 1
* Auteur correspondant
1 Laboratory of Food Biotechnology
Abstract : Aims: To detect and enumerate bifidobacteria in faeces with a new quantitative multiplex real-time PCR (qPCR) method and to compare the results obtained with fluorescence in situ hybridization (FISH) methods. Methods and Results: A multiplex qPCR assay was developed, which enabled the enumeration of Bifidobacterium spp. by targeting the bifidobacterial xylulose- 5-phosphate ⁄ fructose-6-phosphate phosphoketolase gene (xfp) and total bacteria using universal Eub-primers targeting 16S rRNA gene from the domain bacteria. The qPCR assay showed high sensitivity and specificity and a low detection limit of about 2.5 x 10³ bifidobacterial cells per gram of faeces. The qPCR results were compared with FISH combined with microscopy or flow cytometry (FCM). No statistical differences among bifidobacterial counts averages measured in adult faeces with the three methods were observed. Total bacterial count averages were higher with the FISH method coupled with microscopic analyses compared to FISH with FCM, whereas total cell numbers estimated by qPCR were intermediate between the two FISH methods. Conclusions: The new qPCR assay was shown to be sensitive, rapid and accurate for enumerating bifidobacteria in faeces. Significance and Impact of the Study: This method is a valuable alternative for other molecular methods for detecting faecal bifidobacteria, especially when their counts are below the detection limit of the FISH methods.
Keywords : Bifidobacterium spp faeces FISH flow cytometry multiplex qPCR xfp gene
Type de document :
Article dans une revue
Journal of Applied Microbiology, Wiley, 2010, 108 (1), pp.181-193. 〈10.1111/j.1365-2672.2009.04408.x〉
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Contributeur : Gwenaelle Le Blay <>
Soumis le : jeudi 27 janvier 2011 - 15:19:00
Dernière modification le : jeudi 27 janvier 2011 - 16:30:48
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Valentine Cleusix, Christophe Lacroix, G. Dasen, G. Leo, Gwenaëlle Le Blay. Comparative study of a new quantitative real-time PCR targeting the xylulose-5-phosphate/fructose-6-phosphate phosphoketolase bifidobacterial gene (xfp) in faecal samples with two fluorescence in situ hybridization methods. Journal of Applied Microbiology, Wiley, 2010, 108 (1), pp.181-193. 〈10.1111/j.1365-2672.2009.04408.x〉. 〈hal-00560166〉

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