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Standard Operating Procedure (SOP) : 1. General Considerations
Standard Operating Procedure (SOP) : 1. General Considerations
Standard Operating Procedure (SOP) : 1. General Considerations
IS2404-DRB-PCR
IS2404-DRB-PCR and agarose gel-electrophoresis for detection of M. ulcerans DNA
1. General considerations
This document describes the standard operating procedure for IS2404-DRB-PCR and agarose
gel-electrophoresis for detection of M. ulcerans DNA from clinical samples.
Preparation of the dry reagent based (DRB) PCR must be performed in the “Master Mix
laboratory”. This laboratory is exclusively used for this purpose and is free of DNA
amplicons.
The laboratory must be equipped with lab coats, gloves and all necessary laboratory items
exclusively used in this room.
2. DRB-PCR
2.1. Reagents
- PuReTaq Ready-To-Go PCR Beads (GE Healthcare, Munich, Germany)
- Lyophilised primers MU5 and MU6 (e.g. TibMolBiol, Berlin, Germany)
- Purified distilled water, DNAse free (e.g. Roth, Karlsruhe, Germany)
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2.3. Performance of DRB-PCR
Inhibition controls
To prevent false negative results due to inhibition processes inhibition controls (undiluted and
diluted) are processed in parallel to all diagnostic samples.
For inhibition control reactions 1.25 µl of diagnostic (unknown) DNA or dilution and 1.25 µl
positive control DNA is added to the respective tubes.
*As positive control either an already positively tested patient DNA extract may be used, or
confirmed culture extracts.
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Amplification
After preparation of reaction mixtures all samples are transferred to the thermocycler.
Amplification should be carried out according to the following protocol.
Duration of PCR: 1:30 h
3. Agarose gel-electrophoresis
3.1. General considerations
Agarose gel-electrophoresis is performed in the “Agarose gel-electrophoresis laboratory”.
▲ This laboratory is exclusively used for gel-electrophoresis and must not be used for
DNA extraction, master mix preparation or DRB-PCR!
The laboratory must be equipped with lab coats, gloves and all necessary laboratory items
exclusively used in this room. No change of material/equipment between this room and other
rooms is allowed.
3.2. Reagents
- Agarose low EEO (standard agarose, e.g. Applichem)
- 10x TBE buffer (e.g. Roth)
- GelRedTM (Biotium, Hayward, CA)
- 10x loading dye Bluejuice (e.g. Invitrogen, Darmstadt, Germany)*
- DNA ladder, 100 bp (e.g. Invitrogen)
*Loading dye can be alternatively prepared by mixing glycerol (50 %) with
Bromophenolblue (0.25 %) and distilled water (49.75%).
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3.3. Materials and instruments
- Lab coat
- Gloves (disposable, nitrile)
- Pipettes (0,1-10 µl, 2-20 µl)
- DNase-free Pipette tips (10 µl, 20 µl)
- DNase-free reaction tubes 1,5 ml,
- Reaction tube rack
- Erlenmeyer flask, plastic
- Water resistant pen
- UV-Light Photo documentation System
- Electorophoresis chamber & Power supply
- Microwave oven
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Result of sample Result of inhibition control Interpretation of results
Negative Positive Sample negative
Positive Positive Sample positive
Negative Negative Sample inibite*
Positive Negative Sample positive
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Protocol: DRB-PCR for molecular confirmation of suspected BUD patients
Date……....……....…..
No. Patient´s samples Dilution No. Inhibition control to No. Positive- and negative
corresponding controls
patients’ samples
1 10 E 11 1 21 Negative extraction control
2 10 E 12 2 22 Positive PCR control
3 10 E 13 3 23 Negative PCR control
4 10 E 14 4 24
5 10 E 15 5 25
6 10 E 16 6
7 10 E 17 7
8 10 E 18 8
9 10 E 19 9
10 10 E 20 10
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4. External quality assurance
External quality assurance is carried out at DITM/LMU laboratories. In accordance with
different phases of the PCR installation at INH, specimens are initially tested at INH and
DITM/LMU in parallel, see SOP S1 “Collection, transport and storage of diagnostic
specimens”. If results obtained at INH are throughout concordant with results of DITM/LMU
the final phase starts. Then, aliquots of all extracts (25 µl for swabs and punch biopsy
extracts, 10 µl for FNA extracts) are sent quarterly to DITM/LMU by means of DHL courier
service for external quality assurance.
5. References
Stinear, T., B. C. Ross, J. K. Davies, L. Marino, R. M. Robins-Browne, F. Oppedisano, A.
Sievers, and P. D. Johnson. 1999. Identification and characterization of IS2404 and IS2606:
two distinct repeated sequences for detection of Mycobacterium ulcerans by PCR. J Clin
Microbiol 37:1018-23.