Techne PrimeQ - Manual (Page 144)

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3.16 Analysis method: Plus-minus scoring
This type of assay is used to record the presence or absence of a PCR product. Input data can
either be kinetic (whereby the number of readings is >1) or end-point (a reading taken at the end
of the run). The assay uses fluorophore chemistry in the same way as in the other assays such
that the fluorescence produced during the reaction will be proportional to the number of target
DNA molecules.
Plus-minus assays can be performed with or without the use of an internal positive control (IPC),
although its use can prove helpful to ensure that a failed PCR is not mistaken for a negative result.
A no template control (NTC) is used to set confidence thresholds above which all unknowns will be
scored as positive and below which they will be scored as negative. Samples between the two
threshold values are scored as undetermined. With no user input, the software will automatically
analyse the raw data (or baseline corrected data) to measure the difference in fluorescence
between the unknown samples and NTC.
3.16.1 Assay requirements
• Need at least one reporter dye.
• Need at least two cycles in the stage.
• Need a dye defined as a PRD for PRD correction options to be available.
• Need at least two readings for baseline correction to be available.
• Need a no template control (NTC).
3.16.2 Setup
• In the Analysis Selection box, highlight the stage name for analysis to be applied and click
Edit. The Analysis Wizard will launch.
• Select Plus-Minus Scoring from the drop-down menu in the Analysis Method selection
box. Assign dye usage and click Next. The Plus-Minus Wizard will launch.
• If a PRD was assigned in the dye usage menu, the next screen will offer the option of PRD
correction. Click Next.
Raw data
for reporter
with or
without PRD
correction
Baseline
correction
(where >1
reading)
Select
reading(s)
for analysis
Set confidence
threshold
Report Plus/minus
score
Raw data
for reporter
with or
without PRD
correction
Baseline
correction
(where >1
reading)
Select
reading(s)
for analysis
Set confidence
threshold
Report Plus/minus
score