Development and optimization of an in-house PCR method for molecular diagnosis of pertussis

• Published in: Mikrobiyoloji bülteni Vol. 45; no. 4; p. 632
• Main Authors: Güldemir, Dilek, Akbaş, Efsun, Nar Ötgün, Selin, Tekin, Alicem, Esen, Berrin
• Format: Journal Article
• Language: Turkish
• Published: Turkey 01.10.2011
• Subjects: Bordetella pertussis - genetics; Bordetella pertussis - isolation & purification; DNA Primers - standards; DNA Transposable Elements - genetics; DNA, Bacterial - isolation & purification; Humans; Pertussis Toxin - genetics; Pharynx - microbiology; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; Sensitivity and Specificity; Whooping Cough - diagnosis; Whooping Cough - microbiology
• ISSN: 0374-9096

Pertussis (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 inhouse 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 inhouse 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 103 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.