Protocol for homogenisation of Cystic fibrosis (CF) sputum samples in preparation for Aspergillus fungal DNA extraction
Procedure for the homogenization of CF sputum samples prior to DNA extraction:
1) Collect or transfer the sputum sample into a sterile 50mL plastic centrifuge tube.
2) Add an equal volume of Sputasol (dithiothreitol 1.4%) (Oxoid Ltd) to the sputum sample.
Shake the mixture well then incubate at 37?C for 30 minutes with periodic shaking.
3) Sonicate for 120 seconds at an amplitude of 295 µm.
a. Place the plastic centrifuge tube on ice in a polystyrene box (to prevent heat
transfer during sonication).
b. Use plastic tubes only (glass may break during sonication).
c. In order to achieve a sonication amplitude of 295µm, the volume of sample to be
sonicated should first be determined and the correct sonication microtip,
recommended by the manufacturer, should be used. The output is then adjusted
to achieve the amplitude.
? d. Any sonicator can be used but for this procedure a SonicsVC505 ultrasonic
processor was used in a sound abating enclosure. 3 mm stepped microtips were
used with a four element coupler to process up to four samples simultaneously.
This size of microtip will process samples from 0.5 to 10mL in volume. A 40%
output applied to a 3 mm microtip, in a four element coupler, will produces an
amplitude of 295 µm . Sonication is performed continuously for 120 seconds.
4) Heat the sample at 80?C for 20 minutes (ensures mycobacterial decontamination) then
allow to cool [This step only necessary if clinical suspicion of tuberculosis]
5) Perform MycXtra Fungal DNA extraction protocol, as per manufacturer’s instructions -
Procedure for clear free-flowing BAL samples.
This protocol homogensies the sputum sample causing breakdown of sputum mucins and biofilms without disrupting the Aspergillus fungal cell wall or fungal DNA integrity.
This procedure is not recommended for organisms other than Aspergillus as cell wall
integrity may vary in different genera.
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This UDP was kindly supplied by Caroline Baxter, University Hospital of South Manchester Real Time PCR in the Identification and Management of Aspergillus in CF
12211C G Baxter, A M Jones, A K Webb, D W Denning. University Hospital of South
2Manchester, Education and Research Department, Manchester, UK; Manchester
Adult CF Unit, Manchester, UK. Thorax 2010;65:A11-A12. Doi:10.1136/thx.2010.150912.19
Sputasol (dithiothreitol 1.4%) (Oxoid Ltd, Basingstoke, UK). Product code - SR0233A
? SonicsVC505 ultrasonic processor (Sonics and Materials Inc, Newtown, CT, USA)
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