FTD Respiratory pathogens 21 plus - Mikrogen

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9. Additionally required equipment FTD Respiratory pathogens 21 plus version 5/ March 2012 english FTD Respiratory pathogens 21 plus The FTD Resp 21 plus is suited for use with the ABI 7500 (Applied Biosystems ® ), Bio-Rad CFX96 (BIO-RAD), LightCycler ® 480 (Roche) and RotorGene 6000 (Qiagen former Life Science Corbett)/ Rotor-Gene Q (Qiagen). The assay has been fully validated on an ABI 7500 with the AgPath-ID One-Step RT-PCR Kit (Ambion ® ) and with the EasyMAG (bioMérieux). If you want to use different cyclers, enzymes or extraction methods, please firstly check their compatibility with FTD. • Real time PCR master mix and enzyme. We recommend the AgPath-ID One-Step RT-PCR Kit (Ambion ® , cat# 4387391). • Disposable powder-free gloves • Pipettes (adjustable) • Sterile pipette tips with filters • Vortex mixer • Desktop centrifuge • For the ABI 7500, Bio-Rad CFX96 and LightCycler ® 480 96 well PCR plates and plate sealers are recommended. For the usage of the RotorGene 6000/ Rotor-Gene Q it is recommended to use 0.1 ml strip tubes and caps. • Sample rack 10. Samples This test is for use with extracted RNA and DNA from respiratory samples (throat/nasal swabs, bronchoalveolare lavage and sputum) of human origin. In some cases other sample types may be tested including post mortem material such as heart and lung. For long term storage FTD recommends to store all samples at -20°C until extraction. - 10 -
11. Procedure FTD Respiratory pathogens 21 plus version 5/ March 2012 english FTD Respiratory pathogens 21 plus 11.1 Preliminary procedure with using the EasyMAG If you want to use different extraction methods, please firstly check their compatibility with FTD! Extraction of specimens and negative control with the EasyMAG: 1. Thaw one negative control (Resp NC, 330 µl aliquot, white cap) and one internal control (Resp IC, blue cap). Before use, the reagents have to be thawed completely, mixed (by pipetting or short vortexing) and spun down briefly! 2. Extract 4, 6 or 12 samples or multiples of 4, 6, 12 samples and the NC. We recommend a starting volume for the extraction of 300 µl and an elution volume of 75 µl. To increase the sensitivity of the test, you can use a higher starting volume for the extraction of your specimens according to your extraction method i.e. a starting volume of 600 µl and an elution volume of 75 µl. 3. Add 3 µl internal control (IC, blue cap) directly to the lysis buffer of each extraction. Adding the internal control to each of your samples and to the negative control is a very important step to review the nucleic acid isolation has been successful and to check for possible PCR inhibition! 4. Do not extract positive controls! - 11 -
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Page 3 and 4: 1. Identification of the manufactur
Page 7 and 8: 5. Contents FTD Respiratory pathoge
Page 9: 8. Principle of the method FTD Resp
Page 15 and 16: Preparation of a 96 well plate for
Page 17 and 18: 12. Programming of the thermo cycle
Page 25 and 26: 17. Troubleshooting FTD Respiratory
Page 27 and 28: There are only two exceptional case
Page 31 and 32: Pathogen FluA FTD resp+ H1N1 FTD Fl

FTD Respiratory pathogens 21 plus - Mikrogen

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