step-by-step procedure for use of ftd diagnostic assays on ... · eliteingenius –setup manual...
TRANSCRIPT
Step-by-step procedure for use of FTD
diagnostic assays on the ELITe InGenius™ platform (ELITEch Group)
Materials preparation
1. Mastermix:- FTD Enzyme, Buffer and Primer/probe mix are combined in a 2 ml Sarstedt tube
(ref. 72.694.006) according to FTD usual proportions. However, set a final volumefor 2 additional reactions to take into account the dead volume during pipetting bythe instrument (Table 1)
- The Mastermix can be used for up to 3 consecutive runs on the same day ofreconstitution but it needs to be homogenized by short vortexing between eachrun. Mastermix is stored in the cooling block of the instrument at 8°C during all theduration of the experiment following standard recommendations.
- Do not use the Mastermix for more than 1 day and do not store itat -20°C
Table 11 12
PPmix 4.5 µl 21 µl
Buffer 37.5 µl 175 µl
Enzyme 3 µl 14 µl
Total 45 µl 210 µl
FTD-X
Number of reactions
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2. Internal control:- Because 10 µl is the minimal volume that can be pipetted by the ELITe InGenius™,
FTD Internal control pool (IC) is diluted 5 times in TE buffer before the run andaliquoted in the provided 0.5 ml Sartstedt tube (ref. 72.730.005). Set a finalvolume for two additional reactions than requested (Table 2).
- The diluted and aliquoted IC can be stored for several weeks at -20°C. The IC tubeneeds to be changed by a fresh one for every new run to guarantee the stability ofthe IC detection over the different runs.
Table 2
Materials preparation
Number of
reactionsFTD IC pool (µl) TE buffer (µl)
1 6 24
2 8 32
3 10 40
4 12 48
5 14 56
6 16 64
7 18 72
8 20 80
9 22 88
10 24 96
11 26 104
12 28 112
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3. Negative & Positive controls:- Negative control (NC) with the ELITe InGenius™ is a « PCR only » control (water
or TE buffer). Pipet 15 µl of this NC in an elution tube of the ELITe InGenius™. 10µl will be used for the real-time PCR. In case you want to run a NC following boththe extraction and real-time PCR processes, pipet the NC provided in FTD kits in asonicator tube and consider this track as a generic sample. The volume of NCshould be the same as your sample(s).
- 15 µl of the FTD Positive control (PC) is also pipetted in an elution tube of theELITe InGenius™.
Materials preparation
4. Samples:- Handle your samples as for an usual ELITe InGenius™ run.
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1. Upload FTD assay parameters on the ELITe InGenius™- Flash the assay and the reagent list for run of a negative control, a positive
control, or a generic sample (a). The digital file for each FTD assay is provided bythe ELITech support group ([email protected]).
- These assays include (b) general settings about pipetting volumes (Mastermix,Eluates, IC, etc.) and the targeted pathogens; (c) PCR control (RT step, cycles,temperature, timing, etc.); and (d) an interpretation model that will help for theanalysis of amplification data and determine if a detection is positive or negative.
ELITe InGenius™ run
a b
c d
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2. Run set-up- Choose the appropriate name of the FTD assay for each track of your ELITe
InGenius™ run (negative control, positive control or generic) (a)
- Enter lot numbers of the negative control (water or TE buffer) and positivecontrols used (b)
- Enter sample ID (c)
- Place the Mastermix and IC tubes at the appropriate positions in the cooling blockof the ELITe InGenius™ (d)
ELITe InGenius™ run
a b
c d
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- Follow the usual steps for set-up of an experiment on the ELITe InGenius™
platform
- Start the run
ELITe InGenius™ run
a b c
d e f
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3. Data export and analysis- Approve the results for the tracks with NC and PC (a)
- Print pdf report (b)
- Export raw data
- Analyze the data with your usual criteria
ELITe InGenius™ run
a
b
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If you have any further questions or suggestions,
please contact [email protected]
or check the FAQ section on our website
www.fast-trackdiagnostics.com