Development of multiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pneumonia
Establishing a microbial diagnosis for patients with community-acquired pneumonia (CAP) is still challenging and is often achieved in only 30-50% of cases. Polymerase chain reaction (PCR) has been shown to be more sensitive than conventional microbiological methods and it could help to increase t...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Malaysian Society of Parasitology and Tropical Medicine
2011
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| Online Access: | http://irep.iium.edu.my/10716/ http://irep.iium.edu.my/10716/ http://irep.iium.edu.my/10716/1/545_-_556_Farah_Al-Marzooq.pdf |
| Summary: | Establishing a microbial diagnosis for patients with community-acquired pneumonia
(CAP) is still challenging and is often achieved in only 30-50% of cases. Polymerase chain
reaction (PCR) has been shown to be more sensitive than conventional microbiological
methods and it could help to increase the microbial yield for CAP patients. This study was
designed to develop, optimize and evaluate multiplex real-time PCR as a method for rapid
differential detection of five bacterial causes of CAP namely Streptococcus pneumoniae,
Burkholderia pseudomallei and atypical bacterial pathogens, Mycoplasma pneumoniae,
Chlamydophila pneumoniae and Legionella pneumophila. Duplex and triplex real-time PCR
assays were developed using five sets of primers and probes that were designed based on an
appropriate specific gene for each of the above CAP pathogens. The performance of primers
for each organism was tested using SYBR Green melt curve analysis following monoplex realtime
PCR amplification. Monoplex real-time PCR assays were also used to optimize each
primers-probe set before combining them in multiplex assays. Two multiplex real-time PCR
assays were then optimized; duplex assay for the differential detection of S. pneumoniae and
B. pseudomallei, and triplex assay for the atypical bacterial pathogens. Both duplex and
triplex real-time PCR assays were tested for specificity by using DNA extracted from 26
related microorganisms and sensitivity by running serial dilutions of positive control DNAs.
The developed multiplex real-time PCR assays shall be used later for directly identifying CAP
causative agents in clinical samples.
INTRODUCTION
The etiologic diagnosis of CAP remains an
uneasy task, with the causative organisms
often identified in only up to 50% of cases.
This is mainly due to difficulties in culturing
and to the delayed results associated with
conventional methods (serology and culture),
which often allow a retrospective diagnosis
only (Chan et al., 2007; Nolte, 2008).
Molecular methods such as PCR offer
a better approach for the rapid diagnosis
of CAP (Benson et al., 2008). Several
conventional PCR assays have been
developed for each individual respiratory
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