DNA Preparation for the MycoTOOL Mycoplasma Real-Time …€¦ · Both manual sample preparation kits follow the same workflow based on the same principle of precipitation of nucleic

For use in quality control/manufacturing process only.

Roche CustomBiotech provides innovative solutions for mycoplasma testing, including the MycoTOOL Mycoplasma Real-Time PCR Kit, which is used for quality control of select biopharmaceuticals. The kit has been validated according to European Pharmacopoeia (EP) chapter 2.6.7. guidelines for validation of nucleic acid amplification techniques (NAT) for mycoplasma detection.

Roche offers multiple DNA purification methods, and evalua-ted three of them in comparison to the validated method for preparing DNA for testing with the MycoTOOL Mycoplasma Real-Time PCR Kit. Results show that all three methods perform comparably for detection of many regulatory recommended mycoplasma species.

Purification methodsThree Roche DNA purification methods were evaluated for efficiency in preparing samples for PCR analysis with the MycoTOOL Mycoplasma Real-Time PCR Kit.A

utom

ated

Man

ual

MagNA Pure 96 system (Validated method)

Full validation report available under Confidential Disclosure Agreement. Summary available at http://go.roche.com/mycotool_poster

MagNA Pure 24 system

QC Sample Preparation Kit

DNA preparation for the MycoTOOL Mycoplasma Real-Time PCR Kit detection of regulatory recommended mycoplasma speciesEvaluation of three DNA purification methods

Please contact your local CustomBiotech representative

Europe, Middle East, Africa, Latin AmericaPhone +49 621 759 8580Fax +49 621 759 [email protected]

United States Phone +1 800 428 5433 (toll-free)Fax +1 317 521 4065 [email protected]

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Published byRoche Diagnostics GmbH Sandhofer Straße 11668305 MannheimGermany

© 2021 Roche Diagnostics GmbHAll rights reserved.

Japan Phone +81 3 6634 1046Fax +81 3 5479 [email protected]

Asia Pacific Phone +65 6371 6638Fax +65 6371 6601 [email protected]

custombiotech.roche.com/mycotool

1) According to the MycoTOOL Mycoplasma Real-Time PCR Kit Instructions for Use, a biological sampleis regarded as positive if at least one of four technical replicates yields a positive signal. The recoverycontrol was checked for amplification only if the biological sample was negative for mycoplasma.Three biological samples were evaluated for each concentration.

2) Negative control showed a positive signal due to end-user pipetting error.

The three manual purification methods perform com parably. All six species were detected in both concen trations. The three methods can be recommended as a DNA isolation and puri fication method for the MycoTOOL Mycoplasma Real-Time PCR Kit. Product-specific validation is required.

Results and summary1 (Data on file at Roche)

Method evaluation workflow

Sample

ALL eluates: Detection

with LightCycler®

480 II Real-Time

PCR system and

MycoTOOL Mycoplasma

Real-Time PCR Kit

DNA elution in 200 μL



(Validated method)

Automated extraction from 1 mL sample with the MagNA Pure 96 system

or

Automated extraction from 1 mL sample with the MagNA Pure 24 system

or

Manual extraction from 1 mL sample with the QC Sample Preparation Kit

1 3DNA purification2 PCR analysis4

* Standards recommended by EP 2.6.7. were used: A. laidlawii, M. arginini, M. fermentans, M. pneumoniae, M. hyorhinis, M. orale.Source: Mycoplasma Biosafety Service, Vienna, Austria.

Notes • CHO cells were kept frozen until use. • DNA extraction with the MagNA Pure 96 system (Automated Sample Preparation), and the QC Sample Preparation Kit (Manual Sample Preparation)

was performed according to the MycoTOOL Mycoplasma Real-Time PCR Kit Instructions for Use v9. DNA extraction with the MagNA Pure 24 system was performed according to the Instructions for Use of the Pathogen Universal Protocol.

Infection of 5 mL CHO cells

with two concentrations of each inactivated

mycoplasma standard (100 and 10 CFU/mL;

6 different mycoplasma standards*)

Standard Purification Method 100 CFU/mL 10 CFU/mL

A. laidlawii

MagNA Pure 96 system Detected Detected


QC Sample Preparation Kit Detected Detected

M. arginini




M. fermentans




M. pneumoniae




M. hyorhinis

MagNA Pure 96 system2 Detected Detected



M. orale




Regulatory disclaimer

For use in quality control/manufacturing process only.

MycoTOOL Mycoplasma Real-Time PCR Kit: For customers in the European Economic Area: Contains SVHC: octyl/ nonylphenol ethoxylated. For use in analytical activities such as monitoring and quality control and under controlled conditions only – acc. to Art. 56.3 and 3.23 REACH Regulation.

The LightCycler® 480 system is for life science research use only. Not for use in diagnostic procedures.

MYCOTOOL, MAGNA PURE, LC and LIGHTCYCLER are trademarks of Roche. Other brands or product names are trademarks of their respective holders.

Documents

DNA Preparation for the MycoTOOL Mycoplasma Real-Time …€¦ · Both manual sample preparation kits follow the same workflow based on the same principle of precipitation of nucleic