Development of a multiplex PCR assay for detection and differentiation of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica in Nasopharyngeal specimens

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Development of a multiplex PCR assay for detection and differentiation of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica in Nasopharyngeal specimens

 

Author: Cheung, Kit-sum Caren
Title: Development of a multiplex PCR assay for detection and differentiation of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica in Nasopharyngeal specimens
Degree: M.Sc.
Year: 2006
Subject: Hong Kong Polytechnic University -- Dissertations.
Whooping cough -- Diagnosis.
Polymerase chain reaction.
Bordetella.
Department: Dept. of Health Technology and Informatics
Pages: x, 51 leaves : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2174228
URI: http://theses.lib.polyu.edu.hk/handle/200/2684
Abstract: Whooping cough is one of the most common causes of death resulted from infectious disease worldwide before implementation of vaccination. Despite a high vaccine uptake, resurgences of this disease have been reported in certain developed countries. Bordetella (B.) pertussis and B. parapertussis are etiologic agents for the disease of whooping cough. Apart from these two species, B. bronchiseptica, another member of the family, is an opportunistic pathogen for humans. A multiplex PCR assay was designed in this study; this assay employed three published primer pairs targeting IS 481, IS 1001 and Flagellin A genes of B. pertussis, B. parapertussis and B. bronchiseptica, respectively. These primers enabled simultaneous detection and differentiation of the three Bordetella species. The detection limit of the multiplex PCR was 1.3 CFU/PCR for B. pertussis, 13 CFU/PCR for B. parapertussis and 130 CFU/PCR for B. bronchiseptica. The multiplex PCR was tested against 86 nasopharyngeal swabs from patients with suspected pertussis. Thirty eight samples were positive with the IS 481 sequence; B. pertussis was isolated from 11 of these 38 samples. The rest of the PCR positive samples had no pathogen isolated. Inhibitor was not detected in the PCR negative samples. When compared with a modified gold standard established in this study, the sensitivity was 29% and 100% for culture and multiplex PCR, respectively. The specificity was 100% for both culture and multiplex PCR. The multiplex PCR designed in this study was found to be a rapid, sensitive and specific for detection of B. pertussis, B. parapertussis and B. bronchiseptica. Such assay can be adopted by clinical laboratories as an alternative means for diagnosis of whooping cough.

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