title: Optimized PCR-Temporal Temperature Gel Electrophoresis compared to cultivation to assess diversity of gut microbiota in neonates.
abstract: Temporal Temperature Gel Electrophoresis of amplified 16S rRNA gene sequences (16S rDNA PCR-TTGE) constitutes a culture-independent molecular method used to study bacterial communities. All the technical steps are crucial for quality and exhaustiveness of the results obtained by such approach. Careful optimization of the protocols used is ideally needed for each ecosystem studied. We present here the strategy used to construct an optimized protocol for a 16S rDNA PCR-TTGE-based analysis of gut microflora in neonates. Improvement of the different steps, i.e. total DNA extraction, amplification in terms of efficiency and reduction of heteroduplex formation, TTGE migration conditions and bacterial identification from TTGE patterns, was performed. The optimized protocol was used for the subsequent analysis of 14 stool samples comparatively to a culture-based method. We showed that a specifically designed ladder representative of the diversity of the studied microflora is a useful tool for the identification of bacterial taxa despite biases inherent to 16S rRNA genes, including intra-genomic heterogeneity. Cultivation and PCR-TTGE gave congruent results but cultivation was more efficient for the detection of minor populations whereas PCR-TTGE gave a more complete description of the major populations. Finally, we demonstrated the reliability, the detection sensitivity and the convenience of the optimized 16S rDNA PCR-TTGE method compared with cultural approaches for studying the premature neonate gut microbiota.
authors.affiliation: Université Montpellier 1, Laboratoire de Bactériologie-Virologie, EA 3755 UM1, Faculté de Pharmacie, 15 Avenue Charles Flahault, Montpellier Cedex 5, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Roudière
authors.forename: Aurélien
authors.initials: A
authors.lastname: Jacquot
authors.forename: Hélène
authors.initials: H
authors.lastname: Marchandin
authors.forename: Fabien
authors.initials: F
authors.lastname: Aujoulat
authors.forename: Raymonde
authors.initials: R
authors.lastname: Devine
authors.forename: Isabelle
authors.initials: I
authors.lastname: Zorgniotti
authors.forename: Hélène
authors.initials: H
authors.lastname: Jean-Pierre
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Picaud
authors.forename: Estelle
authors.initials: E
authors.lastname: Jumas-Bilak
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods*; D044822:Biodiversity; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005243:Feces / Q000382:microbiology; D005260:Female; D041981:Gastrointestinal Tract / Q000382:microbiology*; D006358:Hot Temperature; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S