Comparison of real-time PCR with SYBR Green I or 5'-nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria.
PCR primers and hybridization probes were designed for the 16S rRNA genes of six bacterial species or groups typically present in human faeces or used in the dairy industry. The primers and probes were applied for quantification of the target bacterial genomes added in artificial DNA mixtures or faecal DNA preparations, using dot-blot hybridization and real-time PCR with SYBR Green I and TaqMan chemistries. Dot-blot hybridization with (33)P-labelled oligonucleotide probes was shown to detect a 10 % target DNA fraction present in mixed DNA samples. Applicability of the rDNA-targeted oligonucleotide probes without pre-enrichment of the 16S gene pool by PCR was thus limited to the detection of the predominant microbial groups. Real-time PCR was performed using a 96-well format and was therefore feasible for straightforward analysis of large sample amounts. Both chemistries tested could detect and quantify a subpopulation of 0.01 % from the estimated number of total bacterial genomes present in a population sample. The linear range of amplification varied between three and five orders of magnitude for the specific target genome while the efficiency of amplification for the individual PCR assays was between 88.3 and 104 %. Use of a thermally activated polymerase was required with the SYBR Green I chemistry to obtain a similar sensitivity level to the TaqMan chemistry. In comparison to dot-blot hybridization, real-time PCR was easier and faster to perform and also proved to have a superior sensitivity. The results suggest that real-time PCR has a great potential for analysis of the faecal microflora.
Malinen
Erja
E
University of Helsinki, Faculty of Veterinary Medicine, Department of Basic Veterinary Sciences, Section of Microbiology, PO Box 57, FIN-00014 Helsinki University, Finland.
Kassinen
Anna
A
Rinttilä
Teemu
T
Palva
Airi
A
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D052160:Benzothiazoles; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D003959:Diamines; D005243:Feces / Q000382:microbiology*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D019914:Taq Polymerase / Q000378:metabolism
D052160:Benzothiazoles; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D003959:Diamines; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D009930:Organic Chemicals; D011804:Quinolines; D012336:RNA, Ribosomal, 16S; C098022:SYBR Green I; D019914:Taq Polymerase