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Journal of Clinical Microbiology, June 2005, p. 2724-2728, Vol. 43, No. 6
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.6.2724-2728.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Novel Approach for Assessing Performance of PCR Cyclers Used for Diagnostic Testing

D. Schoder,¹ A. Schmalwieser,² G. Schauberger,² J. Hoorfar,^3* M. Kuhn,⁴ and M. Wagner¹

Institute for Milk Hygiene, Milk Technology and Food Science,¹ Institute for Medical Physics and Biostatistics, Veterinary University, Veterinärplatz 1, 1210 Vienna, Austria,² Danish Institute for Food and Veterinary Research, Bülowsvej 27, DK-1790 Copenhagen, Denmark,³ Congen Biotechnologie, Robert-Roessler Strasse 10, 13125 Berlin, Germany⁴

Received 11 November 2004/ Accepted 15 January 2005

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 µl of distilled water at 13 different block positions. The certified temperature accuracy of the measurement system was ±0.3°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°C, whereas less-accurate cyclers failed to reach the set temperature by 13 to 20°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.

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* Corresponding author. Mailing address: Danish Institute for Food and Veterinary Research, Bülowsvej 27, DK-1790 Copenhagen V, Denmark. Phone: 45 72346251. Fax: 45 72346001. E-mail: jho{at}dfvf.dk.

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Journal of Clinical Microbiology, June 2005, p. 2724-2728, Vol. 43, No. 6
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.6.2724-2728.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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