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Addition of a Homologous Internal Control to a Real-Time PCR Assay for Detection of Bordetella pertussis

¹ Molecular Diagnostics Laboratory, Institute of Hygiene, Medical University of Graz, Graz, Austria;² Institute of Laboratory Medicine, General Hospital Linz, Linz, Austria;³ Roche Applied Science, Vienna, Austria;

^aaddress correspondence to this author at: Molecular Diagnostics Laboratory, Institute of Hygiene, Medical University of Graz, Universitätsplatz 4, A-8010 Graz, Austria; fax 43-316-380-9649, e-mail harald.kessler@meduni-graz.at

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Pertussis, also called whooping cough, is caused by Bordetella pertussis. The disease may show an atypical course, particularly in neonates and elderly patients. A rapid and safe diagnostic method is thus essential for appropriate treatment and prophylaxis. Culture has been considered the gold standard for detection of B. pertussis, but this method often lacks sensitivity, and a minimum of 4 days may be required to obtain results (1)(2). PCR is a rapid, sensitive, and specific method for the diagnosis of pertussis (3)(4)(5).

In this study, a new molecular assay was established based on real-time PCR and including a homologous internal control (IC). We evaluated the performance of this assay with a commercially available genomic DNA isolate and with clinical samples.

The new molecular assay consisted of a protocol for manual extraction of DNA followed by generation of the amplification product by real-time PCR. The assay was based on the amplification of a 181-bp fragment of the repetitive insertion sequence IS481, which has been described in B. pertussis and Bordetella holmesii and may be present in Bordetella bronchiseptica (6)(7)(8)(9)(10) (see Table 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue12). We determined assay linearity and detection limit by analyzing 10-fold dilutions of the ATCC genomic DNA isolate 9797D from B. pertussis. Interassay variation was determined with 7 dilutions of the genomic DNA isolate (5 determinations on 5 different days), whereas intraassay variation was determined with 3 samples (5 determinations within a single assay). All assays for determination of inter- and intraassay . . . [Full Text of this Article]

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				C. Koidl, M. Bozic, A. Burmeister, M. Hess, E. Marth, and H. H. Kessler Detection and Differentiation of Bordetella spp. by Real-Time PCR J. Clin. Microbiol., February 1, 2007; 45(2): 347 - 350. [Abstract] [Full Text] [PDF]