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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...

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Main Authors: Mahmud, Mohammed Imad A., Al-Marzooq, Farah, How, Soon Hin
Format: Article
Language:English
Published: Malaysian Society of Parasitology and Tropical Medicine 2011
Subjects:
QR Microbiology
R Medicine (General)
Online Access:http://irep.iium.edu.my/10716/1/545_-_556_Farah_Al-Marzooq.pdf
http://irep.iium.edu.my/10716/
http://www.msptm.org/journal.html
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spelling my.iium.irep.107162012-01-30T01:03:46Z http://irep.iium.edu.my/10716/ Development of multiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pneumonia Mahmud, Mohammed Imad A. Al-Marzooq, Farah How, Soon Hin QR Microbiology R Medicine (General) 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 pa Malaysian Society of Parasitology and Tropical Medicine 2011-12 Article REM application/pdf en http://irep.iium.edu.my/10716/1/545_-_556_Farah_Al-Marzooq.pdf Mahmud, Mohammed Imad A. and Al-Marzooq, Farah and How, Soon Hin (2011) Development of multiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pneumonia. Tropical Biomedicine, Vol 28 (3). pp. 545-556. ISSN 0127-5720, OCLC: 12391449 http://www.msptm.org/journal.html
institution Universiti Islam Antarabangsa Malaysia
building IIUM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider International Islamic University Malaysia
content_source IIUM Repository (IREP)
url_provider http://irep.iium.edu.my/
language English
topic QR Microbiology
R Medicine (General)
spellingShingle QR Microbiology
R Medicine (General)
Mahmud, Mohammed Imad A.
Al-Marzooq, Farah
How, Soon Hin
Development of multiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pneumonia
description 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 pa
format Article
author Mahmud, Mohammed Imad A.
Al-Marzooq, Farah
How, Soon Hin
author_facet Mahmud, Mohammed Imad A.
Al-Marzooq, Farah
How, Soon Hin
author_sort Mahmud, Mohammed Imad A.
title Development of multiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pneumonia
title_short Development of multiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pneumonia
title_full Development of multiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pneumonia
title_fullStr Development of multiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pneumonia
title_full_unstemmed Development of multiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pneumonia
title_sort development of multiplex real-time pcr for the rapid detection of five bacterial causes of community acquired pneumonia
publisher Malaysian Society of Parasitology and Tropical Medicine
publishDate 2011
url http://irep.iium.edu.my/10716/1/545_-_556_Farah_Al-Marzooq.pdf
http://irep.iium.edu.my/10716/
http://www.msptm.org/journal.html
_version_ 1643606404208001024
score 13.211869

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