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Performance of an automated platform

Abstract number: O177

Sahli R., Jaton K., Andre C., Meylan P., Telenti A., Bille J., Greub G.

Objectives: Polymerase chain reaction (PCR) is commonly used to detect microbial nucleic acids in clinical samples. However, significant risk of PCR contamination, high reagents costs, and significant workload precluded the wide use of PCR for the aetiological diagnosis of infectious diseases. We thus established a high throughput versatile automated molecular platform and analysed its performance including the critical aspects of troubleshooting.

Methods: DNA and RNA were extracted with MagNApure (Roche) and EasyMAG (BioMérieux), respectively. RNA was converted to cDNA prior to PCR. A TECAN liquid handling system (LHS) was used for 384 well PCR plate setup. Real-time PCR was performed with an ABI 7900HT.

Results: A total of 5953 analyses and 3037 runs have been done during the study period. The median time from reception of the sample to results was overall of 28.3 hours. Time to results were significantly longer when the pathogen to be detected was a RNA virus (because of the retrotranscription step), a fungus (lysis step before DNA extraction) or a CMV/EBV quantitative PCR (runs performed only three time a week; p < 0.001). Longer time to results were also due to invalid runs, with need of repetition of the PCR reactions in a subsequent run: the median time to results was 27 hours for 5097 analyses that did not need to be repeated, 54 hours for 646 analyses repeated twice and 100 hours for 154 analyses repeated three times. Among all 3037 runs, 264 runs were invalid (8.7%). Runs were mainly considered invalid because of the negativity of the ten copies positive control (7.1% of a total of 3037 runs). Major causes of invalid runs were hardware problems with the liquid handling system (2.3% of all runs), use of a defective real time PCR master mix (1.8%), human procedural errors (1.8%), progressive degradation of the positive control (1.4%) and defective primer and probes (0.49%). Contaminations were detected in only 21 runs (0.7%).

Conclusions: Our platform allows to perform a comprehensive set of microbiological diagnostic tests with a high success rate (>91%). Improvements in terms of time to result and reduction of repeats are being addressed thanks to this evaluation.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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