Addition of a Homologous Internal Control to a Real-Time PCR Assay for Detection of Bordetella pertussis
| Autor: | Harald H. Kessler, Christoph Koidl, Jörg Berg, Egon Marth, Markus Stöcher, Michael Bozic, Gerhard Mühlbauer |
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| Rok vydání: | 2005 |
| Předmět: |
Adult
DNA Bacterial Male Bordetella pertussis Time Factors Adolescent Serial dilution Whooping Cough Clinical Biochemistry Polymerase Chain Reaction law.invention law medicine Humans Child Whooping cough Polymerase chain reaction Aged Aged 80 and over Bordetella holmesii Bordetella bronchiseptica biology Biochemistry (medical) Infant Middle Aged Reference Standards biology.organism_classification medicine.disease Molecular biology genomic DNA Real-time polymerase chain reaction Child Preschool Female |
| Zdroj: | Clinical Chemistry. 51:2404-2406 |
| ISSN: | 1530-8561 0009-9147 |
| Popis: | 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 variation included negative … |
| Databáze: | OpenAIRE |
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