Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2011 May;70(1):1-9.
doi: 10.1016/j.diagmicrobio.2010.11.014. Epub 2011 Mar 11.

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

Kathleen A Thurman  1 , Agnes K Warner, Kelley C Cowart, Alvaro J Benitez, Jonas M Winchell
Affiliations
Comparative Study

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

Kathleen A Thurman et al. Diagn Microbiol Infect Dis. 2011 May.

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.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
(A–B) Analysis of CPC and no template control in triplicate using the multiplex assay on the ABI 7500.
Fig. 2
Fig. 2
(A–D) Analysis of representative clinical specimens for MP181 (A), CP-Arg (B), Pan-Leg (C), and a negative specimen (D), using the multiplex assay on the ABI 7500.
Fig. 3
Fig. 3
Comparison of multiplex versus singleplex performance.
See this image and copyright information in PMC

References

    1. Apfalter P., Barousch W., Nehr M., Makristathis A., Willinger B., Rotter M., Hirschi A.M. Comparison of a new quantitative ompA-based real-time PCR TaqMan assay for detection of Chlamydia pneumoniae DNA in respiratory specimens with four conventional PCR assays. J. Clin. Microbiol. 2003;41:592. - PMC - PubMed
    1. Bartlett J.G., Mundy L.M. Community-acquired pneumonia. N. Engl. J. Med. 1995;333:1618–1624. - PubMed
    1. Birkeback N.H., Jensen J.S., Seefeldt T., Degn J., Huniche B., Andersen P.L., Ostergaard L. Chlamydia pneumoniae infection in adults with chronic cough compared to healthy blood donors. Eur. Respir. J. 2000;16:108–111. - PubMed
    1. Cunha B.A. The atypical pneumonias: clinical diagnosis and importance. Clin. Microbiol. Infect. 2006;12:12–24. - PMC - PubMed
    1. Dowell S.F., Peeling R.W., Boman J., Carlone G.M., Fields B.S., Guarner J., Hammerschlag M.R., Jackson L.A., Kuo C.C., Maass M., Messmer T.O., Talkington D.F., Tondella M.L., Zaki S.R. Standardizing Chlamydia pneumoniae assays: recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada) Clin. Infect. Dis. 2001;33:492–503. - PubMed

MeSH terms