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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
ReportPlus/minus
score
Raw data
for reporter
with or
without PRD
correction
Baseline
correction
(where >1
reading)
Select
reading(s)
for analysis
Set confidence
threshold
ReportPlus/minus
score
- OPERATOR’S MANUAL 1
- How to use this guide 3
- Contents 7
- 1 Introduction 13
- 1.1 PrimeQ 15
- 1.2 Quansoft 15
- 1.2.3 Program Editor 16
- 1.2.4 PrimeQ reports 16
- 1.2.5 Data Analysis 16
- 1.3 Unpacking 17
- 1.4 Warning! 17
- 1.5 Uses of PrimeQ 17
- 1.6 Plates 17
- 1.7 Thermal seal 18
- 1.8 Contacting us 18
- 1.9 Installing the hardware 18
- 1.9.3 Operator safety 20
- 1.9.4 Installation 20
- 1.10 Before switching on 23
- 1.11 Technical Specification 24
- 1.12 Working conditions 25
- 1.13 Guarantee 25
- WARRANTY 26
- IF BROKEN 26
- 1.14.4 Software upgrades 29
- 1.15.1 Function keys 30
- 1.15.2 Power up screen 30
- 1.15.3 Beeper 30
- 1.15.4 Before you start 31
- 1.15.5 Start-up procedure 31
- 1.16.8 Points to remember 35
- 1.18 LED power settings 37
- 1.19 Inserting a plate 37
- 1.20 After use 38
- 2 Getting started 39
- 2.1 Introduction to PrimeQ 41
- 2.2.1 PCR 42
- 2.3.1 Intercalating dyes 44
- 2.3.3 Molecular Beacons 45
- 2.4 System overview 46
- 2.5 Optics module 46
- 2.6 Thermal cycling module 47
- 2.7 Scanning module 48
- 3 Quansoft 49
- 3.1 Introducing Quansoft 51
- 3.2 Software overview 52
- 3.2.1 Home page 53
- 3.2.2 The Editors 53
- 3.2.2.3 Program Editor 54
- 3.2.2.4 Results Editor 54
- 3.3 Using Quansoft 55
- 3.3.2 Main screen 56
- 3.3.3 Navigation bar 56
- 3.3.4 Title bar functions 57
- 3.3.5 Menu bar functions 58
- 3.3.6 Accessing the editors 59
- 3.4.1 An Overview 60
- 3.4.3 Setting up a program 62
- Comment [MO1]: 65
- Discussed between 65
- Matt and Christian 65
- 3.5 Running an experiment 86
- 3.5.2 Monitoring the run 89
- 3.6 LED intensity settings 92
- 3.7 Results Editor 93
- 3.7.3 Editing a graph 96
- 3.7.5 Log/Audit trail 99
- 3.7.6 Report 99
- 3.8.1 Exporting 101
- 3.8.2 Printing 102
- Data analysis 103
- 3.9 Introduction 105
- 3.9.3 Fit points 106
- -1/slope 107
- 3.11 Analysis method: None 111
- 3.11.2 PrimeQ Report 112
- 3.13.1 Assay requirements 114
- 3.13.2 Setup 114
- 3.13.3 Viewing the results 116
- 3.13.4 PrimeQ Report 118
- 3.14.1 Assay requirements 119
- 3.14.2 Setup 119
- Crossing line 121
- Noise threshold 121
- 3.14.3 Viewing the results 123
- 3.14.4 PrimeQ Report 124
- 3.14.5 Using Cq values 124
- R² = 0.996 E = 1.977 126
- Dissociation curve 134
- 3.15.1 Assay requirements 135
- 3.15.2 Setup 135
- 3.15.3 Viewing the results 141
- 3.15.4 PrimeQ Report 142
- 3.16.1 Assay requirements 144
- 3.16.2 Setup 144
- 3.16.3 Viewing the results 146
- The plus-minus graph 147
- Results table 147
- 3.16.4 PrimeQ Report 148
- 3.17.1 Assay requirements 150
- 3.17.2 Setup 150
- 3.17.3 Viewing the results 152
- Reporter Selection 153
- 3.17.4 PrimeQ Report 155
- 3.18.1 Assay requirements 157
- 3.18.2 Setup 157
- 3.18.3 Viewing the results 158
- Multi-read graph 159
- 3.18.4 PrimeQ Report 160
- 3.19.1 Multiplex setup 162
- 4 Technical information 165
- 4.1.1 Cleaning PrimeQ 167
- 4.1.2 Fuses 167
- 4.1.3 Insulation Testing 168
- 4.1.4 Mantenimiento 168
- 4.2.1 LED 169
- 4.2.2 Filters 169
- 4.2.3 Replacing the fuses 169
- 4.5 Accessories 170
- 4.6 Replacement parts 171
- 4.8 Packaging 174
- 4.9 Trademarks 174
- 5 Troubleshooting 175
- 5.1 Troubleshooting 177
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