Millipore Technical Publications |
 | PO- PS2956EN00 | |
Rapid Mycoplasma Detection Coupling a Novel Sample Preparation Device to Molecular Methods | ||
| Lit No: | PS2956EN00 | |
| Year: | 2009 | |
| Contamination of a bioreactor can lead to significant loss of time, materials and revenue unless identified early in the process. In biopharmaceutical production, one of the primary reasons for batch failure is due to microbial contamination. Conventional culture based methods are of limited use for in-process detection of contamination events due to their long time to result. Contamination with Mycoplasma is of particular concern due to their ability to pass through a sterilizing 0.2 μm filter, and the length of the 28 day compendial test for Mycoplasma detection. There are significant benefits to the availability of faster methods that will provide equivalent or better results. Here, we present an approach to couple membrane filtration based concentration and capture of organisms from complex liquid samples with purification and detection of the organism’s nucleic acid using molecular methods. Using a novel sample preparation device, up to 20 mL of sample containing up to 2 x 108 mammalian cells can be filtered, allowing the testing of volumes equivalent to those used for culture based assays. Mycoplasma is captured with a 0.1 μm membrane, cells are lysed and nucleic acids recovered directly in the device. We have coupled sample preparation with detection of target nucleic acid using an innovative Target Capture and Real-Time Transcription Mediated Amplification method (Real-Time TMA; Gen-Probe Incorporated). Currently, we have developed a process monitoring application to determine the presence or absence of Mycoplasma species with a sensitivity comparable to culture methods (1-10 cfu/mL) in approximately four hours starting from sample filtration to final signal-detection. *Real-Time Transcription-Mediated Amplification is a technology of Gen-Probe Incorporated. | |
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