title: Development of multiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pneumonia.
abstract: 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.
authors.affiliation: Department of Basic Medical Sciences, Kulliyyah of Medicine, International Islamic University Malaysia.
authors.forename: Farah
authors.initials: F
authors.lastname: Al-Marzooq
authors.forename: M A Mustafa
authors.initials: MA
authors.lastname: Imad
authors.forename: S H
authors.initials: SH
authors.lastname: How
authors.forename: Y C
authors.initials: YC
authors.lastname: Kuan
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D016957:Burkholderia pseudomallei / Q000235:genetics / Q000302:isolation & purification; D016993:Chlamydophila pneumoniae / Q000235:genetics / Q000302:isolation & purification; D017714:Community-Acquired Infections / Q000175:diagnosis*; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D016952:Legionella pneumophila / Q000235:genetics / Q000302:isolation & purification; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009177:Mycoplasma pneumoniae / Q000235:genetics / Q000302:isolation & purification; D015345:Oligonucleotide Probes / Q000235:genetics; D018410:Pneumonia, Bacterial / Q000175:diagnosis*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes