title: 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.
abstract: OBJECTIVE
  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.
  
  
  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 10(3) 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.
  
  
  CONCLUSIONS
  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.
authors: Institute of Food Science and Nutrition, Swiss Federal Institute of Technology, Zentrum, Zürich, Switzerland.
authors: V
authors: V
authors: Cleusix
authors: C
authors: C
authors: Lacroix
authors: G
authors: G
authors: Dasen
authors: M
authors: M
authors: Leo
authors: G
authors: G
authors: Le Blay
mesh_terms: D000328:Adult; D000446:Aldehyde-Lyases / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001644:Bifidobacterium / Q000201:enzymology / Q000235:genetics*; D005243:Feces / Q000382:microbiology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007223:Infant; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins; D000446:Aldehyde-Lyases; C065556:fructose-6-phosphate phosphoketolase