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Protocol for homogenisation of Cystic fibrosis (CF) sputum samples

By Ernest West,2014-04-08 21:12
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Protocol for homogenisation of Cystic fibrosis (CF) sputum samples

    Laboratory Protocol

    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.

    NB

    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.

    References

    February 2011

    Page 1 of 2

    Laboratory Protocol

    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)

    February 2011

    Page 2 of 2

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