Quantitative analysis of 16S rDNA using competitive PCR and the QPCR System 5000.
A method for precise and accurate quantification of 16S rDNA has been developed that uses competitive PCR and the QPCR System 5000. The method is based on co-amplification of 16S rDNA sequences, along with an internal standard sequence, using only one set of conserved eubacterial primers. Co-amplified PCR products are rapidly identified and quantified by measuring the electrochemiluminescent signals from specific oligonucleotide reporter probes that are directed against a hypervariable 16S rDNA sequence. Because in the exponential phase of amplification the different target sequences and the internal standard sequence are amplified with the same efficiency, unknown amounts of a target sequence in a sample can be inferred by extrapolating against a standard curve that is generated for the internal standard sequence. This method provides a rapid, nonradioactive and reliable way to simultaneously quantify different specific 16S rDNA targets that are present in low numbers, and may thus be suitable for enumeration of specific target microorganisms in environmental samples.
Blok
H J
HJ
Wageningen Agricultural University, The Netherlands.
Gohlke
A M
AM
Akkermans
A D
AD
D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis*; D004926:Escherichia coli / Q000737:chemistry; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D008163:Luminescent Measurements; D016133:Polymerase Chain Reaction / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D005456:Fluorescent Dyes; D012336:RNA, Ribosomal, 16S