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Hygiene Screening System

- Hybridization Probes (LC 2.0) Catalog No. R 300 31 PCR system for the qualitative detection of Staphylococcus, Micrococcus, and Corynebacterium DNA including the simultaneous identification of Staphylococcus aureus and Micrococcus luteus using the LightCycler 2.0 Carousel-Based System.

Background Information BIOTECON Diagnostics Hygiene Screening System was developed to meet the need for a fast and specific method for monitoring in the pharmaceutical industry. A monitoring study with several pharmaceutical companies revealed that up to 80% of the microorganisms isolated from samples derived from the pharmaceutical environment (personnel, air and surface) belong to the genera Staphylococcus, Micrococcus and Corynebacterium. The method is based on fluorescence-coupled PCR technology (polymerase chain reaction) and by using different fluorescence channels, a stepwise assignment to either one or other of the genus Staphylococcus, Micrococcus and Corynebacterium, is possible within a single PCR reaction. Since 2004, the usage of more sensitive and specific tests has been recommended by the European Pharmacopoeia (E.P., Chapters 5.1.6 and 2.6.1) and by the FDA Aseptic Guideline with reference to molecular biological methods. There is a need for new methods because most of the conventional methods are time-consuming and sometimes show an insufficient accuracy. The Hygiene Screening System achieves these recommendations by providing an innovative method which allows fast and accurate identification.

Analytical Procedure The Hygiene Screening System is designed for the rapid identification of microorganisms of the genera Corynebacterium, Staphylococcus, Macrococcus, Micrococcus, Kocuria, and Kytococcus. Furthermore, some coryneform bacteria and Nesterenkonia halobia are detected. The amplification and detection is optimized for the LightCycler 2.0 Carousel-Based System (Roche Diagnostics). The system uses highly specific HybProbe probes for the simultaneous detection of all the above mentioned genera within 90 minutes. In addition, the most important species and species groups within these genera can be identified via melting curve analysis. Bacterial colonies growing on agar plates are prepared using the Suspension Buffer (Cat. No. SB 200 10). This buffer allows the preparation of target DNA templates for PCR in less than 5 minutes. The Hygiene Screening System provides primers and probes (for sequent specific detection), conveniently premixed. To ensure maximum reliability, the kit contains a positive and a negative control as run controls, as well as an Internal Control (IC) to prevent false negative results due to inhibition of the amplification. The Hygiene Screening System does not include Taq polymerase. The Enzyme Solution supplied by BIOTECON Diagnostics (Cat. No. A 500 01) contains the Taq Polymerase and UNG which prevents carry-over contamination due to old amplificates.

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98,20%

99% 90,20% 83,30%

Hygiene Screening System

Conventional system

Fig. 1: Qualitative Detection with the Hygiene Screening System.

Fig. 2: Hygiene Screening System vs. Conventional Methods.

Specificity and Reproducibility In summer 2007 a ring trial with the Hygiene Screening System was carried out by eight big pharmaceutical companies. The Hygiene Screening System was compared to conventional routine methods and the reproducibility of the system was tested. By using the Hygiene Screening System 99% of the organisms could be detected. By using conventional methods 90.2% of the positive samples and only 83.3% of the negative samples were correctly classified by the participants. This shows that with the Hygiene Screening System a new level of accuracy can be reached. By testing the reproducibility and precision of the Hygiene Screening System was tested with ten target organisms during three days. Again 99% of the tested organisms were correctly detected with the Hygiene Screening System. None of the ring trial participants had worked with the Hygiene Screening System before; although the users had nearly no experience the results produced were excellent. This underlines the high routine suitability of the Hygiene Screening System.

Kit Content Hygiene Screening System Master Mix Hygiene Screening System Internal Control Hygiene Screening System Control Template H2O, PCR-grade

Amount per Kit 3 x 520 l 3 x 32 l 1 x 50 l 1 x 1 ml

Amount per Reaction/Run 16 l 1 l 2.5 l (positive control) 2.5 l (negative control)

License Notice The purchase of this product includes a limited, non-transferable license under U.S. Patents Nos. 6,245,514 and 6,174,670, and corresponding patents and patent applications outside the United States, owned by the University of Utah Research Foundation and licensed by Idaho Technology, Inc. and Roche Diagnostics GmbH, to use only this amount of product for FRET assays solely for food analysis or GMO analysis. No right is conveyed, expressly, by implication or estoppel, for any other patent, such as under any patent for an apparatus or system, or to use this product for any other purpose. The product is covered in-part by US 5,871,908, co-exclusively licensed by Roche Diagnostics GmbH from Evotec OAI AG. foodproof is a registered trademark of BIOTECON Diagnostics. Lightcycler is a trademark of Roche Diagnostics.

BCD_PS_R30031_V02/08

www.bc-diagnostics.com