MycoTOOL. A PCR-based Mycoplasma Detection Kit
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1 MycoTOOL A PCR-based Mycoplasma Detection Kit
2 Release Testing with the MycoTOOL Test is Approved by the EMA (European Medicines Agency) for Several Pharmaceutical Products Mycoplasms are frequent causes of contamination in bio pharmaceutical production, cell therapy and tissue engineering. Traditional detection methods, required by Pharmacopoeias and drug regulating agencies worldwide, use growth on culture media to detect contaminating organisms. These culture-based methods are time-consuming, requiring as much as 28 days to complete, making them laborious and difficult to interpret. Rapid methods, like the new MycoTOOL Test, for detecting Mycoplasma contamination, help to improve efficiency, quality and safety in the manufacturing of pharmaceutical and biological products. Approvals with MycoTOOL Test In July 2009, the European Medicines Agency (EMA) approved release testing with the MycoTOOL Test for a pharmaceutical product from Roche. The Ministry of Health, Labour and Welfare of Japan (MHLW), and the EMA approved the same test concept for pharmaceutical product testing at a global pharma ceutical manufacturer. Cone dryer for large-scale GMP production Results in hours instead of days For life science research only. Not for use in diagnostics procedures. Cellular samples as well as noncellular samples are tested in less than 5 hours. 1 ml Sample or DNA precipitation 450 μl 200 μl eluate 4 x 20 μl 2x Mycoplasma PCR 2x Host-cell PCR GAPDH Results on gel Mycoplasma PCR GAPDH PCR Cellular Cell-free + MycoTOOL Carrier DNA DNA precipitation 450 μl 200 μl eluate 4 x 20 μl 2x Mycoplasma PCR 2x Host-cell PCR GAPDH
3 The Significance of Using Highly Purified Reagents As an integrated pharmaceutical and diagnostic company, Roche has developed a novel set of of reagents, guaranteed to be nucleic acid free based on Roche s ultra-sensitive Quality Control procedures. This technology breakthrough was essential for developing the MycoTOOL PCR Test, making release testing for Mycoplasms more sensitive and reliable than current homebrew PCR tests. Validation at 1 CFU/ml This validation for a CHO cell line was generated using more than 17,000 PCR reactions. Nine isolates were validated at 1 CFU/ml at a 95% confidence level ( 23 out of 24 positive). A tenfold dilution series of 1000 CFU/ml to 0.01 CFU/ml was performed. Each replicate was done in quadruplicate PCR, and 96 PCR reactions were performed per concentration. The established sensitivity is 1 CFU/ml. Increased sensitivity A bridging study comparing the Roche homebrew to the MycoTOOL PCR Test demonstrates higher sensitivity. Validated: 23 out of 24 replicates to be positive Reduced risk of false positives Roche homebrew Species ATCC Sensitivity MycoTOOL Sensitivity A. laidlawii M. hominis A comparison using Roche reagents, and oligonucleotides from a different supplier, clearly shows the risk posed by contaminating DNA. Validated: 23 out of 24 replicates to be positive Species ATCC Claimedsensitivity Probit analysis of validation data 1 CFU/ml CFU/ml A. laidlawii* <1.0 M. arginini M. fermentans M. hominis* <1.0 M. hyorhinis M. orale M. pneumoniae M. salivarium S. citri M. gallisepticum not yet validated M. synoviae not yet validated *for 1 CFU/ml 24 out of 24 positive; 0.5 CFU/ml and 0.1 CFU/ml not tested PCR with purified Roche reagents J No Mycoplasma contamination J High PCR efficiency PCR with purified non-roche reagents J Mycoplasma contamination causes a false positive result J Primer dimer formation causes J lower PCR efficiency
4 Ideal for a Broad Variety of Sample Materials Not validated Validated Cellular matrices 10 CFU/ ml A. laidlawii M. orale 3 CFU/ml 3 CFU/ ml 1 CFU/ ml Roche homebrew Specificity MycoTOOL Specificity MycoTOOL MRC 5 cells Vero cells BHK cells Human stem cells Cell-free matrices Allantoic fluid MycoTOOL Human stem cell MycoTOOL CHO cell Carrier DNA supernatant Buffer These MycoTOOL Test results demonstrate there is no PCR inhibition. Mycoplasms are also easily detected in cell-free matrices with the same sensitivity when MycoTOOL Carrier DNA is added during sample preparation. Species ATCC L. acidophilus 4356 n.d n.d S. bovis n.d n.d. C. sporogenes n.d n.d. n.d. not done MycoTOOL Test specificity is higher than that of generic reagents used for the Roche homebrew. A tenfold increase is shown for L. acidophilus and C. sporogenes. There is no cross-reactivity below 10 4 CFU/ml. Lysis and inhibition control of matrix using parallel GAPDH PCR The risk of obtaining false negatives is of parti cular importance, as undetected contamination can have negative health consequences. False negatives can be produced by inefficient cell lysis, matrix inhibition, or lack of sensitivity that is a function of the sample preparation reagents. If the positive control is limited to using a plasmid, a false negative result cannot be detected. For this reason, it is important to monitor the efficiency of the sample preparation and the lysis of host cells using parallel matrix specific PCR. The MycoTOOL Test performs a parallel PCR by detecting the GAPDH gene in host cells. If amplification of the GAPDH gene is not appropriate for your application, Roche Custom Biotech can adapt the MycoTOOL Test to your requirements. No carryover contamination The MycoTOOL Test uses dutp instead of dttp for incorporation into the PCR product. When dutp-containing amplification product contaminates the test workflow, it will be digested using the enzyme uracil-dna glycosylase (UNG) instead of being reamplified in the subsequent PCR reaction. A Mycoplasma sequence with a deletion is used to code for the positive control. When the amplifica tion product of this plasmid contaminates the PCR, it can easily be differentiated from a real Mycoplasma contamination by its shorter length. Change control notification The design and the production process for the MycoTOOL PCR Test are controlled by the change control notification procedures of the Roche Quality Control system, ensuring that the product will retain its proven high quality.
5 A Test Accepted for Validation According to European Pharmacopeia High sensitivity Validation at 1 CFU/ml at a 95% confidence level ( 23 out of 24 positive) Limit of detection (LOD) <1 CFU/ml for most isolates Compatible with a diverse spectrum of sample types Cellular matrices: Human cells (primary and continuous), canine cells, nonhuman primate cells, many different rodent cell types Cell-free matrices: Culture supernatants of CHO or human stem cells, egg derived samples Detects the broad panel of Mollicute species Detection predicted for over 90 species due to universal primer design Including Mycoplasma, Spiroplasma, and Acholeplasma Minimizes risk of false negatives and false positives Lysis control of matrix eliminates risk of undetected intracellular Mycoplasma Positive control verifies potential PCR inhibition Nucleic acid free reagents, as tested using Roche's ultra-sensitive Quality Control procedures, prevent false positives Use of uracil-dna glycosylase minimizes the risk of PCR carryover contamination Ensures safety and process quality during production Change control procedures are in place for all reagents and kits All critical reagents are GMP manufactured For life science research only. Not for use in diagnostics procedures. Trademarks MYCOTOOL is a trademark of Roche. Ohter brands or product names are trademarks of their respective holders. Ordering Information Product Pack Size Catalog Number MycoTOOL PCR Mycoplasma Detection Kit Kit for testing of 10 mammalian cell culture samples (1 ml each) for the absence of Mycoplasms Consists of 2 subkits: MycoTOOL Mycoplasma Detection Prep Kit MycoTOOL Mycoplasma Detection Amplification Kit
6 Published by Roche Diagnostics GmbH Sandhofer Straße Mannheim Germany 2010 Roche Diagnostics. All rights reserved Your Roche Custom Biotech Customer Service Customers in Europe, Middle East, Africa, and Latin America: Roche Diagnostics Deutschland GmbH Sandhofer Straße Mannheim, Germany Phone Fax Customers in the United States: Roche Diagnostics Corporation Roche Applied Science 9115 Hague Road P.O. Box Indianapolis, IN , USA Phone , ext (toll-free) Fax Customers in Canada: Roche Diagnostics 201, Boulevard Armand-Frappier HV 4A2 Laval, Québec, Canada Phone Fax Customers in Japan: Roche Diagnostics K.K. Roche Custom Biotech Applied Science 6-1, Shiba 2-chome Minato-ku, Tokyo , Japan Phone Fax Customers in Asia Pacific: Roche Diagnostics Asia Pacific Pte. Ltd. Regional Sales and Market Development 298 Tiong Bahru Road # 16-01/06 Central Plaza Singapore, Phone Fax custombiotech.roche.com/mycotool
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