Activity and DNA contamination of commercial polymerase chain reaction reagents for the universal 16S rDNA real-time polymerase chain reaction detection of bacterial pathogens in blood.
Universal 16S rRNA gene polymerase chain reaction (PCR) is a promising means of detecting bacteremia. Among other factors, the PCR reagents play a prominent role for obtaining a high sensitivity of detection. The reagents are ideally optimized with respect to the amplifying activity and absence of contaminating DNA. In this study, it was shown in a universal 16S rDNA real-time PCR assay that commercial PCR reagents can vary greatly among each other in these characters. Only 1 of the 5 reagents tested met the criteria of sensitive detection of pathogen DNA with a minimum of false-positive results. The reagent was validated by the detection of pathogens at low titers using bacterial DNA extracted from blood that was spiked with various Gram-positive and Gram-negative bacteria.
Mühl
Helge
H
Institut für Angewandte Mikrobiologie und Biotechnologie, Hochschule Bremerhaven, An der Karlstadt 8, D-27568 Bremerhaven, Germany.
Kochem
Anna-Julia
AJ
Disqué
Claudia
C
Sakka
Samir G
SG
D016470:Bacteremia / Q000097:blood* / Q000382:microbiology*; D052160:Benzothiazoles; D004247:DNA / Q000032:analysis*; D003959:Diamines; D004866:Equipment Contamination; D005189:False Positive Reactions; D005798:Genes, Bacterial; D006090:Gram-Negative Bacteria / Q000235:genetics; D006094:Gram-Positive Bacteria / Q000235:genetics; D006801:Humans; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012336:RNA, Ribosomal, 16S; D011933:Reagent Kits, Diagnostic; C098022:SYBR Green I; D004247:DNA; D019914:Taq Polymerase