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Preperation and extraction of Nucleic Acid
Sample Preparation Methods for Bordetella pertussis Real-Time PCR
M.RIFFELMANN, C H WIRSING VON KOENIG, C.VAHRENHOLZ, J SCHMETZ, C BOCK
Institut für Infektiologie Krefeld GmbH, Institut für Hygiene und Labormedizin, Klinikum Krefeld, Germany.
Abstract:
Background and aims: Pertussis is a re-emerging disease, and its diagnosis is increasingly done by real-time PCR. The effectiveness of commercially available kits for preparing Bordetella DNA from respiratory material has not yet been validated.
Methods: A total of 100 nasopharyngeal swabs, tracheal secretions and other respiratory materials were spiked with various amounts of B.pertussis cells. Preparation methods were based on commercially available ion-exchange chromatography columns. We compared the QIAamp? DNA mini kit, which is validated for DNA extraction from various materials with the QIAamp? Min Elute Virus Vacuum kit, QIAamp? DSP Virus kit and the QIAamp? Virus BioRobot MDx kit, which are validated for preparation of viral RNA and DNA from cell-free material. PCR was done by two real-time PCR’s based on a hybridisation probes format (LightCycler, Roche) and on a TaqMan format (SDS 7700, Applied Biosystems). PCR’s amplified different parts of the insertion sequence IS 481 from B.pertussis.
Results: All four kits tested are useful for preparation of B. pertussis DNA from respiratory materials. The reproducibility of all kits was acceptable. All procedures were linear over a broad range (5-500.000 organisms/PCR). The MinElute Virus Vacuum kit and the DSP Virus kit did not effectively remove inhibitory substances
Conclusions: Commercial nucleic acid preparation systems were effective in preparing DNA for B.pertussis real-time PCR, the QIAamp® DNA Mini Kit may be used for the extraction of single samples, and the QIAamp® Virus BioRobot MDx Kit may be used for preparation of 32-96 samples with the BioRobot MDx
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