Detection of Mycoplasma pneumoniae , Chlamydia pneumoniae , and Legionella spp. in clinical specimens using a single-tube multiplex real-time PCR assay

• Published in: Diagnostic microbiology and infectious disease Vol. 70; no. 1; pp. 1 - 9
• Main Authors: Thurman, Kathleen A, Warner, Agnes K, Cowart, Kelley C, Benitez, Alvaro J, Winchell, Jonas M
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01.05.2011; Elsevier; Elsevier Biomedical
• Subjects: Bacteriological Techniques - methods; Bacteriology; Biological and medical sciences; Chlamydia Infections - diagnosis; Chlamydia Infections - microbiology; Chlamydophila pneumoniae; Chlamydophila pneumoniae - genetics; Chlamydophila pneumoniae - isolation & purification; Community-Acquired Infections - diagnosis; Community-Acquired Infections - microbiology; Community-acquired pneumonia; Fundamental and applied biological sciences. Psychology; Humans; Infectious Disease; Infectious diseases; Internal Medicine; Legionella; Legionella - genetics; Legionella - isolation & purification; Legionellosis - diagnosis; Legionellosis - microbiology; Medical sciences; Microbiology; Miscellaneous; Multiplex real-time PCR; Mycoplasma Infections - diagnosis; Mycoplasma Infections - microbiology; Mycoplasma pneumoniae; Mycoplasma pneumoniae - genetics; Mycoplasma pneumoniae - isolation & purification; Oligonucleotide Probes - genetics; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; Real-time PCR; Ribonuclease P - genetics; Sensitivity and Specificity; Real-time PCR; Multiplex real-time PCR; Community-acquired pneumonia; Lung disease; Chlamydia pneumoniae; Pneumonia; Chlamydiaceae; Legionellaceae; Multiplex polymerase chain reaction; Respiratory disease; Mycoplasma pneumoniae; Legionella; Real time; Mycoplasmataceae; Mollicutes; Polymerase chain reaction; Mycoplasmatales; Community acquired infection; Chlamydiales; Bacteria; Detection; Clinical isolate
• ISSN: 0732-8893; 1879-0070
• DOI: 10.1016/j.diagmicrobio.2010.11.014

Abstract A multiplex real-time PCR assay for the detection of Mycoplasma pneumoniae (MP181), Chlamydia ( Chlamydophila ) pneumoniae (CP-Arg), Legionella spp. (Pan-Leg), and the human RNase P (RNase P) gene was developed for rapid testing of atypical bacterial respiratory pathogens in clinical specimens. This method uses 4 distinct hydrolysis probes to detect 3 leading causes of community-acquired pneumonia. The assay was evaluated for specificity and sensitivity by testing against 35 related organisms, a dilution series of each specific target and 197 clinical specimens. Specificity testing demonstrated no cross-reactivity. A comparison to previously validated singleplex real-time PCR assays for each agent was also performed. The analytical sensitivity for specific pathogen targets in both the singleplex and multiplex was identical (50 fg), while efficiencies ranged from 82% to 97% for the singleplex assays and from 90% to 100% for the multiplex assay. The clinical sensitivity of the multiplex assay was improved for the Pan-Leg and CP-Arg targets when compared to the singleplex. The MP181 assay displayed equivalent performance. This multiplex assay provides an overall improvement in the diagnostic capability for these agents by demonstrating a sensitive, high-throughput and rapid method. This procedure may allow for a practical and efficient means to test respiratory clinical specimens for atypical pneumonia agents in health care settings and facilitate an appropriate public health response to outbreaks.