Utility of real-time amplification of selected 16S rRNA gene sequences as a tool for detection and identification of microbial signatures directly from clinical samples.
The potential of incorporating a real-time PCR for amplification and detection of 16S rRNA gene signatures directly from clinical samples was assessed as a tool for diagnostics. Universal PCR primers spanning short variable regions (~500 bp) were optimized for real-time PCR and tested in comparison with a longer fragment (~1400 bp) generated from block-based amplification. Real-time PCR had improved sensitivity of detection (8% increase), decreased amplification time and simplified downstream processing. The real-time PCR primers also offered an improvement in detection of bacteria from samples that demonstrated inhibition with the block-based primers and in the resolution of mixed-sequence traces. In addition to testing primer sensitivity, the effect of amplifying and sequencing two different variable regions of the 16S rRNA gene on organism identification was compared. By amplifying and sequencing a shorter variable region, the number of species that were identified to the species level was reduced by 18%.
Department of Bioanalysis and Horizon Technologies, Health Protection Agency Microbiology Services Division Colindale, 61 Colindale Avenue, London NW9 5EQ, UK.
Kirstin J
KJ
Edwards
Department of Bioanalysis and Horizon Technologies, Health Protection Agency Microbiology Services Division Colindale, 61 Colindale Avenue, London NW9 5EQ, UK.
Julie M J
JMJ
Logan
Department of Bioanalysis and Horizon Technologies, Health Protection Agency Microbiology Services Division Colindale, 61 Colindale Avenue, London NW9 5EQ, UK.
Sally
S
Langham
Department of Bioanalysis and Horizon Technologies, Health Protection Agency Microbiology Services Division Colindale, 61 Colindale Avenue, London NW9 5EQ, UK.
Craig
C
Swift
Department of Bioanalysis and Horizon Technologies, Health Protection Agency Microbiology Services Division Colindale, 61 Colindale Avenue, London NW9 5EQ, UK.
Saheer E
SE
Gharbia
D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D001424:Bacterial Infections / Q000175:diagnosis*; D001431:Bacteriological Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D020459:Genes, rRNA; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
D017931:DNA Primers; D012336:RNA, Ribosomal, 16S