sybr-green-i and Bacteremia

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.

Topics: Bacteremia; Benzothiazoles; Diamines; DNA; Equipment Contamination; False Positive Reactions; Genes, Bacterial; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Nucleic Acid Denaturation; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Reagent Kits, Diagnostic; Reproducibility of Results; RNA, Ribosomal, 16S; Sensitivity and Specificity; Taq Polymerase

The prevalence of the Panton-Valentine leukocidin (PVL) gene in Staphylococcus aureus was investigated with a simple, reproducible and rapid real-time LightCycler SYBR Green I PCR assay. The PVL gene was detected in one isolate from 65 patients with S. aureus bacteraemia, in four isolates from 55 patients with respiratory tract infections, and in two isolates from 91 patients with cutaneous infections. In contrast, 15 of 25 cutaneous isolates of methicillin-resistant S. aureus (MRSA) were positive. All PVL-positive cutaneous MRSA isolates were community-acquired and comprised three different clones as determined by pulsed-field gel electrophoresis. The PVL gene was detected in isolates from patients with recurrent primary skin infections and S. aureus bacteraemia, but PVL did not seem to be an important virulence factor in the pathogenesis of staphylococcal bacteraemia.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Bacteremia; Bacterial Toxins; Benzothiazoles; Child; Child, Preschool; Diamines; DNA, Bacterial; Electrophoresis, Gel, Pulsed-Field; Exotoxins; Female; Humans; Infant; Infant, Newborn; Leukocidins; Male; Methicillin Resistance; Middle Aged; Organic Chemicals; Polymerase Chain Reaction; Prevalence; Quinolines; Respiratory Tract Infections; Staphylococcal Infections; Staphylococcal Skin Infections; Staphylococcus aureus; Sweden