MycoTOOL A PCR-based Mycoplasma Detection KitMycoTOOL A PCR-based Mycoplasma Detection Kit i t i u y...
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MycoTOOL A PCR-based Mycoplasma Detection Kit distributed by Europe, Middle East, Africa, and Latin America Roche Diagnostics Deutschland GmbH Sandhofer Straße 116 68305 Mannheim, Germany Phone +49 621 759 8580 Fax +49 621 759 8610 [email protected] United States Roche Diagnostics Corporation Roche Applied Science 9115 Hague Road P.O. Box 50414 Indianapolis, IN 46250-0414, USA Phone +1 800 428 5433, ext. 14649 (toll-free) Fax +1 317 521 4065 [email protected] Canada Roche Diagnostics 201, Boulevard Armand-Frappier HV 4A2 Laval, Québec, Canada Phone +1 450 686 7050 Fax +1 450 686 7012 [email protected] Japan Roche Diagnostics K.K. Roche Custom Biotech Applied Science 6-1, Shiba 2-chome Minato-ku, Tokyo 105-0014, Japan Phone +81 3 5443 5285 Fax +81 3 5443 7934 [email protected] Asia Pacific Roche Diagnostics Asia Pacific Pte. Ltd. Regional Sales and Market Development 298 Tiong Bahru Road # 16-01/06 Central Plaza Singapore, 168730 Phone +65 6371 6638 Fax +65 6371 6601 [email protected] Your Roche Custom Biotech Customer Service custombiotech.roche.com/mycotool Published by Roche Diagnostics GmbH Sandhofer Straße 116 68305 Mannheim Germany © 2011 Roche Diagnostics. All rights reserved. 05619378990 0411 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 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 05 200 709 001 Consists of 2 subkits: MycoTOOL Mycoplasma Detection Prep Kit 05 184 592 001 MycoTOOL Mycoplasma Detection Amplification Kit 05 184 240 001 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 A Test Accepted for Validation According to European Pharmacopeia 2.6.7. For use in quality control/manufacturing process only. MYCOTOOL is a trademark of Roche. Other brands or product names are trademarks of their respective holders.
Transcript of MycoTOOL A PCR-based Mycoplasma Detection KitMycoTOOL A PCR-based Mycoplasma Detection Kit i t i u y...
MycoTOOL A PCR-based Mycoplasma Detection Kit
distributed by
Europe, Middle East, Africa, and Latin AmericaRoche Diagnostics Deutschland GmbHSandhofer Straße 11668305 Mannheim, GermanyPhone +49 621 759 8580Fax +49 621 759 [email protected]
United StatesRoche Diagnostics CorporationRoche Applied Science9115 Hague RoadP.O. Box 50414Indianapolis, IN 46250-0414, USAPhone +1 800 428 5433, ext. 14649 (toll-free)Fax +1 317 521 [email protected]
CanadaRoche Diagnostics201, Boulevard Armand-FrappierHV 4A2 Laval, Québec, CanadaPhone +1 450 686 7050Fax +1 450 686 [email protected]
JapanRoche Diagnostics K.K.Roche Custom BiotechApplied Science6-1, Shiba 2-chomeMinato-ku, Tokyo 105-0014, JapanPhone +81 3 5443 5285Fax +81 3 5443 [email protected]
Asia PacificRoche Diagnostics Asia Pacific Pte. Ltd.Regional Sales and Market Development298 Tiong Bahru Road# 16-01/06 Central PlazaSingapore, 168730Phone +65 6371 6638Fax +65 6371 [email protected]
Your Roche Custom Biotech Customer Service
custombiotech.roche.com/mycotool
Published byRoche Diagnostics GmbHSandhofer Straße 11668305 MannheimGermany
© 2011 Roche Diagnostics.
All rights reserved.
05619378990 0411
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 typesCell-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 designIncluding �� Mycoplasma, Spiroplasma, and Acholeplasma
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
05 200 709 001
Consists of 2 subkits:MycoTOOL Mycoplasma Detection Prep Kit 05 184 592 001
MycoTOOL Mycoplasma Detection Amplification Kit 05 184 240 001
Minimizes risk of false negatives and false positives
Lysis control of matrix eliminates risk of ��undetected intracellular MycoplasmaPositive control verifies potential PCR inhibition ��Nucleic acid free reagents, as tested using ��Roche's ultra-sensitive Quality Control procedures, prevent false positivesUse 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 kitsAll critical reagents are GMP manufactured ��
A Test Accepted for Validation According to European Pharmacopeia 2.6.7.
For use in quality control/manufacturing process only.
MYCOTOOL is a trademark of Roche.Other brands or product names are trademarks of their respective holders.
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.
Release Testing with the MycoTOOL Test is Approved by the EMA (European Medicines Agency) for Several Pharmaceutical Products
450 μl
450 μl
DNA precipitation
4 x 20 μl
4 x 20 μl
2x Host-cell PCR – GAPDH
2x Mycoplasma PCR
2x Host-cell PCR – GAPDH
2x Mycoplasma PCR
1 ml Sample
or
Cellular Cell-free + MycoTOOL
Carrier DNA
DNA precipitation
200 μl eluate
200 μl eluate
Results in hours instead of days
Cellular samples as well as noncellular samples are tested in less than 5 hours.
Cone dryer for large-scale GMP production
Mycoplasma PCR GAPDH PCR Results on gel
For use in quality control/manufacturing process only.
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 essen-tial for developing the MycoTOOL PCR Test, making release testing for Mycoplasms more sensitive and reliable than current homebrew PCR tests.
Increased sensitivity
A bridging study comparing the Roche homebrew to the MycoTOOL PCR Test demonstrates higher sensitivity.
Reduced risk of false positives
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 Claimed- sensitivity
Probit analysis of validation data
1 CFU/ml CFU/ml
A. laidlawii* 27556 + <1.0
M. arginini 23838 + 0.15
M. fermentans 19989 + 0.29
M. hominis* 23114 + <1.0
M. hyorhinis 17981 + 0.51
M. orale 23714 + 0.11
M. pneumoniae 15531 + 0.02
M. salivarium 23064 + 1.41
S. citri 27556 + 0.37
M. gallisepticum 15302 + not yet validated
M. synoviae 25204 + 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� No Mycoplasma contamination� High PCR efficiency
PCR with purified non-Roche reagents� Mycoplasma contamination
causes a false positive result� Primer dimer formation causes� lower PCR efficiency
Validated: 23 out of 24 replicates to be positive
Roche homebrew
MycoTOOL
Species ATCC Sensitivity [CFU/ml]
Sensitivity [CFU/ml]
10 5 1 10 5 1
A. laidlawii 27556 + + – + + +
M. hominis 23114 + – – + + +
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.
The Significance of Using Highly Purified Reagents
Release Testing with the MycoTOOL Test is Approved by the EMA (European Medicines Agency) for Several Pharmaceutical Products
Ideal for a Broad Variety of Sample Materials
Validated
Roche homebrew
MycoTOOL
Specificity [CFU/ml]
Specificity [CFU/ml]
Species ATCC 106 105 104103 102 106 105 104 103 102
L. acidophilus
4356 n.d. + + + - n.d. + + - -
S. bovis 35034 - – - – n.d. - - - - n.d.
C. sporogenes
11437 - + - + n.d. - + + - n.d.
n.d. “not done”
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.
Not validated
A. laidlawii M. orale
Cellular matrices 10 CFU/
ml
3 CFU/ml
3 CFU/
ml
1 CFU/
ml
MycoTOOL
MRC 5 cells + + + +
Vero cells + + + +
BHK cells + + + +
Human stem cells + + + +
Cell-free matrices
MycoTOOL
+ MycoTOOL
Carrier DNA
Allantoic fluid + + + +
Human stem cell + + + +
CHO cell
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.
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 104 CFU/ml.
