原创力文档reliability of quantitative real-time pcr for bacterial detection in cystic fibrosis airway specimens可靠性的定量实时pcr在囊性纤维化气道细菌检测标本.pdfVIP
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reliability of quantitative real-time pcr for bacterial detection in cystic fibrosis airway specimens可靠性的定量实时pcr在囊性纤维化气道细菌检测标本
Reliability of Quantitative Real-Time PCR for Bacterial
Detection in Cystic Fibrosis Airway Specimens
1 2 1 1 1
Edith T. Zemanick *, Brandie D. Wagner , Scott D. Sagel , Mark J. Stevens , Frank J. Accurso , J. Kirk
Harris1
1 Department of Pediatrics, University of Colorado Denver, Aurora, Colorado, United States of America, 2 Department of Biostatistics and Informatics, Colorado School of
Public Health, University of Colorado Denver, Aurora, Colorado, United States of America
Abstract
The cystic fibrosis (CF) airway microbiome is complex; polymicrobial infections are common, and the presence of fastidious
bacteria including anaerobes make culture-based diagnosis challenging. Quantitative real-time PCR (qPCR) offers a culture-
independent method for bacterial quantification that may improve diagnosis of CF airway infections; however, the reliability
of qPCR applied to CF airway specimens is unknown. We sought to determine the reliability of nine specific bacterial qPCR
assays (total bacteria, three typical CF pathogens, and five anaerobes) applied to CF airway specimens. Airway and salivary
specimens from clinically stable pediatric CF subjects were collected. Quantitative PCR assay repeatability was determined
using triplicate reactions. Split-sample measurements were performed to measure variability introduced by DNA extraction.
Results from qPCR were compared to standard microbial culture for Pseudomonas aeruginosa, Staphylococcus aureus, and
Haemophilus influenzae, common pathogens in CF. We obtained 84 sputa, 47 oropharyngeal and 27 salivary specimens from
16 pediatric subjects with CF. Quantitative PCR detected bacterial DNA in over 97% of specimens. All qPCR assays were
highly reproducible at quantities $102 rRNA gene copies/reaction with co
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