Mycobacterium Tuberculosis - Diagenode Diagnostics

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DIAGENODE MYCOBACTERIUM TUBERCULOSIS REAL-TIME PCR USER MANUAL
Protocol
A. Procedures to be followed
A.1 Extraction procedure (see point III. Sample Extraction/DNA Isolation)
Perform this procedure on the appropriate level containment facility.
Please follow the following steps:
• Place sample(s) at room temperature.
NB:
It is recommended to manipulate potential infected samples under a safety cabinet.
(Optional) We highly recommend a pre-treatment procedure before the extraction (See point I.
Sample pre-treatment procedure using the diagenode sonication system: the Bioruptor)
• Choose the appropriate extraction method validated for your sample. (See point III Sample
Extraction/DNA Isolation).
Please, follow manufacturer’s instructions to perform extractions.
(Optional) If using DNA extraction & PCR inhibition control Real-Time PCR, add 10 μl of the
Diagenode DNA extraction & inhibition control (DNA virus culture tube) in the extracted volume
(see point II. DNA Extraction & inhibition control and III. Sample Extraction/DNA Isolation).
NB:
The Diagenode DNA extraction & PCR inhibition control Real-Time PCR is provided separately,
ref: DIA-EIC/DNA-50. You can select the dye adapted to your real-time PCR system (see point VI.
Selection of the appropriate dyes).
• When the extraction procedure is finished, prepare the DNA on ice before using it (or at -
20°C if you don’t use it the same day).
• Place the Mycobacterium tuberculosis positive control tube (for long term storage) on
ice.
NB:
If the extracted samples are at room temperature for a long period of time, DNA could be degraded.
If DNA is degraded, the sensitivity of the PCR will decrease!
If the Mycobacterium tuberculosis positive control tube is at room temperature for a long period
of time (> 24 hours), the positive control could be degraded and influence the results obtained!
A.2 PCR procedures
Perform the procedure on the appropriate level containment facility.
Master mix suggested: Quantifast Multiplex + R kit (ref : 204756), Qiagen (Not supplied) (see point
IV. PCR Reaction Set-up).
NB:
Rotor-Gene Multiplex PCR Kit (Qiagen) (Not supplied) for Qiagen Rotor-Gene
LightCycler ® TaqMan ® Master (Roche) (Not supplied) for LightCycler 2.0 (Lc2.0)
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3 e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50 // Mail: info@diagenode.com