title: A single-step polymerase chain reaction for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae.
abstract: OBJECTIVE
  The critically high prevalence of bacterial otitis media worldwide has prompted a proper disease management. While vaccine development for otitis media is promising, the reliable and effective methods for diagnosis of such etiologic agents are of importance.
  
  
  METHODS
  We developed a multiplex polymerase chain reaction assay for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae. Five primer pairs targeting genes fumarate reductase (H. influenzae), outer membrane protein B (M. catarrhalis), major autolysin (S. pneumoniae), capsulation-associated BexA protein (all encapsulated H. influenzae) and 16S rRNA were incorporated in this single-step PCR. Validation of the multiplex PCR was also performed on clinical isolates.
  
  
  RESULTS
  The developed multiplex PCR was highly specific, enabling the detection of the target pathogens in a specific manner, either individually or as a mixture of all target organisms. The assay was also found to be sensitive with the lowest detection limit of 1 ng of bacterial DNA. When applied to clinical isolates from diverse specimen sources, the multiplex PCR developed in this study correctly identified each microorganism individually or in a combination of two or more target organisms. All results matched with conventional culture identification. In addition, the ability of such assay to differentiate H. influenzae encapsulation from the study clinical isolates was 100%.
  
  
  CONCLUSIONS
  Our multiplex PCR provides a rapid and accurate diagnostic tool for detection of the 4 target organisms. Such assay would serve as a useful tool for clinicians and epidemiologists in their efforts to the proper treatment and disease management caused by these organisms.
authors.affiliation: Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand. kunthalertd@yahoo.com
authors.forename: Duangkamol
authors.initials: D
authors.lastname: Kunthalert
authors.forename: Kritsada
authors.initials: K
authors.lastname: Henghiranyawong
authors.forename: Anchalee
authors.initials: A
authors.lastname: Sistayanarain
authors.forename: Krissana
authors.initials: K
authors.lastname: Khoothiam
mesh_terms: D004269:DNA, Bacterial / Q000032:analysis*; D006193:Haemophilus influenzae / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D001936:Moraxella catarrhalis / Q000235:genetics / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction; D010033:Otitis Media / Q000175:diagnosis* / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial