Effect of comprehensive validation of the template isolation procedure on the reliability of bacteraemia detection by a 16S rRNA gene PCR

• Published in: Clinical microbiology and infection Vol. 10; no. 5; pp. 452 - 458
• Main Authors: Heininger, A., Binder, M., Ellinger, A., Pfisterer, J., Botzenhart, K., Unertl, K., Döering, G.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Elsevier Ltd 01.05.2004; Blackwell Science Ltd; Blackwell
• Subjects: 16S rRNA; Bacteraemia; Bacteremia - diagnosis; Bacteremia - microbiology; Bacterial diseases; Bacterial sepsis; Biological and medical sciences; DNA extraction; DNA Primers; DNA, Bacterial - analysis; DNA, Bacterial - isolation & purification; DNA, Ribosomal - genetics; Escherichia coli; Escherichia coli - genetics; Escherichia coli - isolation & purification; Fundamental and applied biological sciences. Psychology; Genes, rRNA; Human bacterial diseases; Humans; Infectious diseases; Medical sciences; Microbiology; PCR; Polymerase Chain Reaction - methods; Reproducibility of Results; RNA, Ribosomal, 16S - genetics; Staphylococcus aureus; Staphylococcus aureus - genetics; Staphylococcus aureus - isolation & purification; 16S rRNA; Bacteraemia; DNA extraction; PCR; Microbiology; Ribosomal DNA; Bacteremia; Infection; Polymerase chain reaction; Bacteriosis; Isolation; Molecular biology; Reliability
• ISSN: 1198-743X; 1469-0691
• DOI: 10.1111/j.1469-0691.2004.00877.x

The influence of the DNA extraction method on the sensitivity and specificity of bacteraemia detection by a 16S rRNA gene PCR assay was investigated. The detection limit of the assay was 5 fg with purified DNA from Escherichia coli or Staphylococcus aureus, corresponding to one bacterial cell. However, with spiked blood samples, the detection limits were 104 and 106 CFU/mL, respectively. The sensitivity of the S. aureus assay was improved to the level of the E. coli test with the addition of proteinase K to the commercial DNA extraction kit protocol. Ten (16.6%) of 60 amplification reactions were positive with templates isolated from sterile blood, while PCR reagent controls were negative, thereby indicating contamination during the DNA extraction process. Blood samples were spiked with serial dilutions of E. coli and S. aureus cells, and six PCR results were obtained from three extractions for each blood sample. A classification threshold system was devised, based on the number of positive reactions for each sample. Samples were deemed positive if at least four positive reactions were recorded, making it possible to avoid false-positive results caused by contamination. These results indicate that a comprehensive validation procedure covering all aspects of the assay, including DNA extraction, can improve considerably the validity of PCR assays for bacteraemia, and is a prerequisite for the meaningful detection of bacteraemia by PCR in the clinical setting.