Development and Optimization of an In-House PCR Method for Molecular Diagnosis of Pertussis

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dc.contributor.authorGuldemir, Dilek
dc.contributor.authorAkbas, Efsun
dc.contributor.authorOtgun, Selin Nar
dc.contributor.authorTekin, Alicem
dc.contributor.authorEsen, Berrin
dc.date.accessioned2024-04-24T17:47:27Z
dc.date.available2024-04-24T17:47:27Z
dc.date.issued2011
dc.departmentDicle Üniversitesien_US
dc.description.abstractPertussis (whooping cough), caused by Bordetella pertussis is a severe, acute contagious disease of the respiratory system and it affects mostly children and also susceptible individuals of all ages. Although the conventional culture method used for diagnosis is highly specific, it has a lower sensitivity. Therefore, there is a need for a sensitive, specific and rapid method for diagnosis of pertussis. Polymerase chain reaction (PCR), introduced recently as a new approach for diagnosis of pertussis, has been shown to be more sensitive than culture method. Pertussis toxin gene (ptxA-Pr), insertion sequence genes (IS481 and IS1001), adenylate cyclase genes and structural porin and flagellin genes were chosen as targets for PCR, in different studies. This study aimed to develop and optimize a diagnostic in-house PCR method by using primers specific for ptxA-Pr and IS481 gene regions. An in-house PCR method was developed by using primer pairs of PTp1/PTp2 specific for ptxA-Pr gene and PIp1/PIp2 specific for IS481 gene and DNAs of various bacterial reference strains. Throat samples obtained from 45 healthy individuals and B.pertussis reference strain with decreasing concentrations were mixed to constitute a group of representative clinical samples and used to test and optimize sensitivity and specificity of the method. The in-house PCR with PTp1/PTp2 primers showed a very high specificity but a low sensitivity with a value of 34.4 cfu/Rm (colony forming unit/reaction mixture). Whereas, the in-house PCR with PIp1/PIp2 primers exhibited a low specificity due to cross-reactivity with B. pertussis and B.bronchiseptica but much higher sensitivity with a value of 1.12 cfu/Rm. The experiments performed with the representative clinical samples yielded similar results. Simultaneously applied cultivation studies indicated the detection limit of the PCR method as 2 x 10(3) cfu/ml. Based on our results, the PCR targeting IS481 gene had high sensitivity while the PCR targeting ptxA-Pr gene had high specificity. It was concluded that, PCR method targeting the IS481 gene might be used for pre-diagnosis and then PCR for ptxA-Pr gene might be applied for the confirmation of B.pertussis in the molecular diagnosis of pertussis.en_US
dc.identifier.endpage645en_US
dc.identifier.issn0374-9096
dc.identifier.issue4en_US
dc.identifier.pmid22090294
dc.identifier.startpage632en_US
dc.identifier.urihttps://hdl.handle.net/11468/22489
dc.identifier.volume45en_US
dc.identifier.wosWOS:000297000100007
dc.identifier.wosqualityQ4
dc.indekslendigikaynakWeb of Science
dc.indekslendigikaynakPubMed
dc.language.isotren_US
dc.publisherAnkara Microbiology Socen_US
dc.relation.ispartofMikrobiyoloji Bulteni
dc.relation.publicationcategoryMakale - Uluslararası Hakemli Dergi - Kurum Öğretim Elemanıen_US
dc.rightsinfo:eu-repo/semantics/closedAccessen_US
dc.subjectBordetella Pertussisen_US
dc.subjectWhooping Coughen_US
dc.subjectMolecular Diagnosisen_US
dc.subjectIn-House Pcren_US
dc.subjectIs481en_US
dc.subjectPtxa-Pren_US
dc.titleDevelopment and Optimization of an In-House PCR Method for Molecular Diagnosis of Pertussisen_US
dc.titleDevelopment and Optimization of an In-House PCR Method for Molecular Diagnosis of Pertussis
dc.typeArticleen_US

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