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Publikationstyp: Zeitschriftenaufsatz
Dokumentart: Originalarbeit

Publikationsjahr: 2005

AutorInnen: Schoder, D; Schmalwieser, A; Schauberger, G; Hoorfar, J; Kuhn, M; Wagner, M

Titel: Novel approach for assessing performance of PCR cyclers used for diagnostic testing.

Quelle: J Clin Microbiol. 2005; 43(6):2724-2728



Autor/innen der Vetmeduni Vienna:

Schauberger Günther
Schmalwieser Alois
Schoder Dagmar
Wagner Martin

Beteiligte Vetmed-Organisationseinheiten
Abteilung für Lebensmittelmikrobiologie
Abteilung für Physiologie und Biophysik


Zugehörige(s) Projekt(e): Food-PCR: Validierung und Standardisierung diagnostischer PCR-Methoden für den Nachweis von lebensmittelpathogenen Mikroorganismen


Abstract:
As part of a large international project for validation and standardization of PCR, the influence of thermocyclers on PCR was tested. Six brand-new, Peltier technology-driven 96-well thermocyclers were subjected to a novel and stringent in-tube (not block) physical testing. The temperature was directly monitored in PCR tubes containing 50 microl of distilled water at 13 different block positions. The certified temperature accuracy of the measurement system was +/-0.3 degrees C. Finally, the results of the physical testing were compared to those of an amplification efficiency study running an in-house PCR assay. The cyclers did not perform within the manufacturer"s specification. Premature timing, under- and overshooting, and spatial variation of heat transfer were found to be the critical factors. The physical testing allowed us to distinguish accurate from less-accurate (2/6) cyclers. The lack of thermal homogeneities became most evident at the denaturation level during the first 15 s. At the time point zero, the accurate cyclers showed temperature deviations of 0.5 to 1.5 degrees C, whereas less-accurate cyclers failed to reach the set temperature by 13 to 20 degrees C. Consequently, the two less-accurate cyclers could not gain positive PCR results by running an in-house PCR assay. However, by modifying the original temperature protocol by increasing the denaturation temperature and time, the amplification efficiency of these two cyclers could be improved significantly. The results have implication for laboratories using diagnostic PCR testing.

Keywords Pubmed: DNA, Bacterial/analysis
DNA, Bacterial/genetics
Hot Temperature
Listeria/genetics
Nucleic Acid Denaturation
Polymerase Chain Reaction/instrumentation*
Polymerase Chain Reaction/methods
Polymerase Chain Reaction/standards
Reproducibility of Results
Sensitivity and Specificity
Temperature


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