iQ-Design Assays
User Guide
Tests for the real-time PCR detection of microorganisms in food and environmental
samplesTable of Contents
Section 1

Introduction ............................................................................................................................................. 1

Section 2

The iQ-Design Assay Technology ........................................................................................................... 1

Section 3

Kit Components ...................................................................................................................................... 1

Section 4

Shelf Life and Storage............................................................................................................................. 2

Section 5

Materials Required but Not Supplied....................................................................................................... 2
Equipment ............................................................................................................................................... 2
Supplies .................................................................................................................................................. 2

Section 6

Safety Precautions and Recommendations for Best Results .................................................................. 2

Section 7

Protocol ................................................................................................................................................... 4
A. Sample Enrichment and DNA Extraction ............................................................................................ 4
B. Real-Time PCR................................................................................................................................... 4
C. Data Analysis...................................................................................................................................... 7

Section 8

Test Performance and Validation ............................................................................................................ 9

Section 9

Revision History ...................................................................................................................................... 9
Appendix — PCR Mix Calculation Guide .............................................................................................. 10

ii | iQ-Design Assays

Section 1 Introduction

Section 1
Abschnitt 1
Abschnitt 2

Introduction
iQ-Design Assays are specially designed for the rapid detection and characterization of microorganisms infood, water, and environmental samples. Using real-time polymerase chain reaction (PCR), specific DNA
sequences for the target microorganisms are amplified and detected simultaneously by means of fluorescentprobes. The intended users of this kit are trained laboratory personnel who are performing tests to detectmicroorganisms in food and environmental samples. The use of this test allows results to be obtained withina few hours following enrichment of a sample.

Section 2
Abschnitt 3

The iQ-Design Assay Technology

Abschnitt 4

The iQ-Design Assay products are available as individual primer-probe mix reagents that are wet-lab
validated for use with Bio-Rad’s iQ Multiplex Powermix amplification reagent. The iQ-Design Assays aredesigned and optimized for the iQ-Check DNA extraction workflows and compatible with Bio-Rad’s CFX96
Touch Deep Well Real-Time PCR Detection System.

Abschnitt 5

The ready-to-use assay contains oligonucleotides (primers and probes) specific for target microorganisms.
PCR is a powerful technique used to generate many copies of target DNA, called amplicons. During thischain reaction, several cycles of heating and cooling lead to exponential accumulation of these amplicons,
making it eventually possible to detect the target DNA. Each cycle starts with the denaturation of the doublestranded DNA at a high temperature. When the temperature decreases, the primers bind to the target region
and are extended by the DNA polymerase. Two amplicons are thus generated from each initial target DNA
fragment.

Abschnitt 6

In real-time PCR, specific probes bind to target DNA and are lysed by the DNA polymerase during extension.
These probes are linked both to a fluorophore and a quencher, which absorbs the light emitted by thefluorophore. Upon lysis of the probe, the fluorophore and quencher are separated so that fluorescence isreleased. In the absence of target DNA, no fluorescence will be detected. If target DNA is present in thesample, as the number of amplicons increases with each round of amplification, fluorescence intensity also
increases. At each PCR cycle, the optical module, or detector, measures this fluorescence. The software
associated with the instrument plots fluorescence intensity against the number of cycles. This method allows
a simple determination of the presence or absence of the target microorganism in a sample.

Section 3
Abschnitt 7
Abschnitt 8

Kit Components
The iQ-Design Assay is a ready-to-use primer-probe mix optimized for use with iQ Multiplex Powermix. Each
kit comes with 0.8 or 1.6 ml of assay mix, sufficient for 100 or 200 reactions, respectively.

iQ-Design Assays | 1

Section 4 Shelf Life and Storage

Section 4
Abschnitt 9

Shelf Life and Storage

Abschnitt 10

iQ-Design Assays are shipped at room temperature. Upon receipt, store product at 4°C (up to 12 months).
For long-term storage up to the expiration date indicated on the vial, store at –20°C.

Abschnitt 11

If storing the vial at –20°C, prevent oligonucleotide degradation by aliquoting the total volume into smaller
volumes to minimize the number of freeze-thaw cycles.

Section 5
Abschnitt 12Materials Required but Not Supplied
Equipment▪

10, 20, 200, and 1,000 μl micropipets▪

Bio-Rad CFX96 Touch Deep Well Real-Time PCR Detection System (catalog #3600037)

Note: We recommend using an uninterrupted power supply (UPS) with the thermal cycler.

Supplies▪

iQ Multiplex Powermix (catalog #17006573, 200 reactions; 17007081, 100 reactions )▪

PCR plates, tubes, sealing tape, and caps▪

Sterile filter tips adaptable to 10, 20, 200, and 1,000 μl micropipets▪

Tips for Combitip pipets or equivalent repeat pipettors; sterile, individually packaged▪

2 and 5 ml sterile test tubes▪

Powder-free gloves▪

Distilled sterile water▪

Bleach, 5%▪

Cleaning agent such as DNA AWAY or RNase AWAY

Section 6
Abschnitt 13

Safety Precautions and Recommendations for Best Results▪

This test must be performed by trained personnel

2 | iQ-Design Assays

Section 6 Safety Precautions and Recommendations for Best Results▪

Samples and enrichment cultures must be handled as potentially infectious material and discarded
according to local rules and regulations▪

All potentially infectious material should be autoclaved before disposal▪

The quality of results depends on strict compliance with Good Laboratory Practices (for example, the EN
ISO 7218 standard), especially concerning PCR-

Never circulate laboratory equipment (pipets, tubes, etc.) from one workstation to another-

Always use a positive control and a negative control for each series of amplification reactions-

Do not use reagents after their expiration date-

Vortex reagents from the kit before using to ensure homogeneity-

Periodically verify the accuracy and precision of pipets, as well as correct functioning of the
instruments-

Change gloves often, especially if you suspect they are contaminated-Clean workspaces periodically with 5% bleach and other decontaminating agents such as DNA
AWAY-

Use powder-free gloves and avoid fingerprints and writing on tube caps. Both will interfere withdata acquisition▪

It is strongly advised to follow the general requirements described in the standard EN ISO 22174:2005(Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the detection offood pathogens — General requirements and definitions)▪

iQ-Design Assays-▪

CFX96 Touch Deep Well Real-Time PCR Detection System-▪

All substances or mixtures in the test kit are classified products, according to the Globally
Harmonized System (GHS). Contact with acids may cause release of toxic gases. No special
precautions are necessary if used correctly. If the product is inhaled, supply fresh air and consulta doctor in case of complaints. After eye contact with the product, rinse opened eye for several
minutes under running water. If the products are swallowed, induce vomiting and call for medical
help

Improper use of the CFX96 Touch Deep Well Real-Time PCR Detection System may cause
personal injury or damage to the instrument. Some components may pose a risk of personal
injury due to excessive heat if improperly handled. For safe use, the CFX96 Touch Deep Well
Real-Time PCR Detection System must be operated only by qualified laboratory personnel who
have been appropriately trained. Servicing of instrument must be performed only by Bio-Rad
Field Service Engineers

For iQ-Design Assays that do not include an internal control, we recommend performance verification
with your matrix prior to analysis. Certain matrices may contain PCR inhibitors that could impede theamplification reaction. To ensure that amplification proceeds properly, run two tests in parallel:- Perform a 1:10 dilution of the target DNA used for the positive control in distilled sterile water- Perform a 1:10 dilution of the same target DNA in a DNA extract prepared after sample
enrichment

iQ-Design Assays | 3

Section 7 Protocol

Important: The matrix sample used for this enrichment must be free of naturally occurring target DNA.
Inhibition of the reaction will show a shift of the amplification curve toward a higher quantification cycle(Cq refers to the cycle at which the amplification curve crosses the threshold) compared to the Cq valueof the first test (partial inhibition), or as signal extinction (complete inhibition). For samples showing
inhibition (partial or complete), repeat the test by performing a 1:10 dilution of DNA extract in sterile
distilled water.

Section 7
Abschnitt 14

Protocol
A. Sample Enrichment and DNA Extraction
The iQ-Design Assays are designed for use with DNA extraction samples obtained from the Standard or
Easy DNA extraction protocols. Refer to the iQ-Design Assay Technical Report for your specific assay to findfurther details on the sample enrichment and DNA extraction protocol.

B. Real-Time PCR
Instrument and software setup
For instrument setup, follow instructions in the CFX96 Touch Deep Well Instruction Manual (Bulletin#10021337). For software setup instructions, see Chapter 3, Performing Runs.

PCR mix preparation
1. Prepare PCR mix containing the amplification solution (iQ Multiplex Powermix) and the fluorescent probemix (iQ-Design Assay Primer-Probe Mix). Volume of PCR mix needed depends on the number ofsamples and controls to be analyzed. At least one positive and one negative control must be included ineach PCR run. Use the pipetting table in the Appendix to find the correct volumes to use for eachreagent.
2. After preparation, the PCR mix (iQ Multiplex Powermix + iQ-Design Assay Primer-Probe Mix) should be
used immediately or within 1 hr, stored at 2–8°C.
3. Pipet 20 μl of PCR mix into each well according to your plate setup.
4. Add 5 μl of DNA extract and negative or positive control. Do not vortex the sample before pipetting.
Hermetically seal the wells of the plate or strips. It is important to avoid bubbles at the bottom of the wellsby pipetting carefully. As an optional step to eliminate any bubbles, centrifuge the sealed PCR plate or
the PCR strips (quick spin).
5. Place the plate or strips in the thermal cycler. Be sure to place the plate with the A1 well at the upper left
corner. Close the reaction module.

Run PCR
1. Open CFX Manager Software to set up the PCR conditions.
2. On the main window toolbar, click User-defined Run Setup.
4 | iQ-Design Assays

Section 7 Protocol

3. Create a new protocol: on the Protocol tab, click Create New to open the Protocol Editor to create a newprotocol (see Figure 1).

4. Select any step in either the graphical or text display — the step becomes highlighted in blue (see Figure2). Click the temperature or dwell time to directly edit the value. Refer to the iQ-Design Assay Technical
Report for the thermal cycling conditions specific to your assay. Click Insert Step or Delete Step to add
or remove a temperature step from the protocol.

Graphical
display

Text display
Protocolediting
controls
Fig. 2. Protocol Editor. The protocol editing controls are on theleft. Both the graphical and text displays can be edited. Step 1
is highlighted and 95°C is selected for editing in the text display.
5. Click Add Plate Read to Step to designate when the fluorescence data will be acquired during theprotocol. Add the Plate Read step after Step 3.

iQ-Design Assays | 5

Section 7 Protocol

6. Click the number of repeats of a GOTO step to change the number of cycles in the protocol. Click the
GOTO step number to change the steps included in the GOTO loop following the number of cycles in
Figure 3.

Fig. 3. An example of an iQ-Design
Assay thermal cycling condition.
7. Once the protocol is set up, click OK and save the current protocol with the name specific to youriQ-Design Assay.
8. On the Plate Tab, select Create New to open the Plate Editor window (see Figure 4).
9. Click Select Fluorophores and indicate the fluorophores of interest (see Figure 5). Refer to theiQ-Design Assay Technical Report for the fluorophore type specific to your assay.

Fluorophoreand plate
editing
controls

Fig. 5. Plate Editor. The fluorophore and plate editing controls are on the right.
10. Indicate which wells of the plate are used in your experiment, and whether they contain unknown
samples, the positive controls, or the negative controls. Check the fluorophore box under Target Names.
11. Target names and sample names can also be added at this step.
6 | iQ-Design Assays

Section 7 Protocol

12. Select Start Run in the Run Setup window to start the PCR run.
13. Run an existing protocol/plate: Click Select Existing from the Run Setup window to launch the filebrowser and load the previous saved files. Select Start Run to start the PCR run.
Refer to the CFX96 Touch Deep Well Instruction Manual for additional information.

C. Data Analysis
Data can be analyzed directly at the end of the PCR run or later by opening the stored data file. Follow
instructions in the CFX96 Touch Deep Well Instruction Manual for opening data files and setting the data
analysis parameters.

Interpreting results
Once the data analysis parameters have been set, results are interpreted by analyzing the Cq values of eachsample.
Controls▪

Verify the positive and negative controls before interpreting sample results.
-

Negative Control: The negative control monitors contamination. To perform a negative control,
add distilled sterile water in place of the sample to be tested.

-

Positive Control: The positive control confirms that the experimental conditions are met for
identifying a sample containing the target pathogen. A positive control is included with certain iQDesign Assays and is recommended to use in your analysis. For iQ-Design Assays that do notinclude a positive control, a positive control will need to be created:
To create a positive control, use target DNA (synthetic or isolated from a microorganism) inplace of the sample. Adjust the DNA concentration of your positive control solution so that its
amplification curve crosses the threshold after 18–32 PCR cycles (18 ≤ Cq ≤ 32).
When working with a positive control prepared from a microbial culture, a minimum dilution of 2–
3 logs is necessary.
To prepare the dilution add 100 µl of DNA extract obtained from an overnight culture of a target
strain in 9.9 ml of distilled sterile water (2 log). If the Cq of the sample obtained is too low (Cq ≤18), dilute it further by taking 1 ml of this dilution and adding it to 9 ml of sterile distilled water(3 log). If the Cq of the sample is still too high (Cq ≥ 32), use the undiluted sample.
Note: Use caution when working with highly concentrated DNA samples, as they can easily
contaminate your work area and can then be difficult to eliminate.

iQ-Design Assays | 7

Section 7 Protocol

For the experiment to be valid, the controls must have the following results, as summarized in the tablebelow. Otherwise, the PCR reaction must be repeated.
Target*Internal Control Detection**

Negative control

Cq = N/A

Cq ≥ 25

Positive control

18 ≤ Cq ≤ 35

Not significant*For duplex or multiplex assays, there will be multiple target channels. Each target channel will need to meet the interpretation
requirements listed in the table.
**For assays that do not include an internal control, refer to Section 6, Safety Precautions and Recommendations for Best Results, forperformance verification with your specific matrix before analysis to ensure that amplification proceeds properly. A Cq value that is N/Ain the target channel is a negative result.

Refer to Section 6, Safety Precautions and Recommendations for Best Results, for recommended
procedures for positive and negative control result interpretation.
Samples
A positive iQ-Design Assay sample must have a Cq value ≥10 for the target.
▪

If the Cq is <10, verify that the raw data curve is a regular amplification curve (with a flat base line,
followed by a rapid exponential increase of fluorescence and then a flattening out). If the curve seems
correct, it may be considered a positive sample.

▪

If there is no Cq value (Cq = N/A) for the target and the internal control has a Cq ≥25, the sample isconsidered a negative sample.

▪

If the Cq value for the internal control is <25, it is not possible to interpret the result. Verify that thethreshold was correctly placed, or that the curve as raw data is a regular amplification curve. If the curve
does not have a characteristic shape, it will be necessary to repeat the PCR test.

For the experiment to be valid, the samples must have the following results, as summarized in the tablebelow. Otherwise, the PCR reaction must be repeated.

Target*Internal Control Detection**Interpretation

Cq ≥ 10

Not significant

Positive

Cq = N/A

Cq ≥ 25

Negative

Cq = N/A

Cq = N/A

Inhibition* For duplex or multiplex assays, there will be multiple target channels. Each target channel will need to meet the interpretation
requirements listed in the table.
**For assays that do not include an internal control, refer to Section 6, Safety Precautions and Recommendations for Best Results, forperformance verification with your specific matrix before analysis to ensure that amplification proceeds properly. A Cq value that is N/Ain the target channel is a negative result.

8 | iQ-Design Assays

Section 8 Test Performance and Validation

Section 8
Abschnitt 15
Abschnitt 16

Test Performance and Validation
All iQ-Design Assays are developed and optimized following strict guidelines on specificity and sensitivity.
Their performance is wet-lab validated for the following:
Amplification efficiency
Linear dynamic range

90–110 %Minimum of six orders of magnitude

R2

>0.98

Section 9
Abschnitt 17

Revision History
Release date
May 2021
August 2021

Document number
10000142603 Ver A
10000142603 Ver B

ChangeNew document
Addition of legal disclaimer

iQ-Design Assays | 9

Appendix — PCR Mix Calculation Guide Revision History

Appendix — PCR Mix Calculation Guide
To find the correct volumes to use when preparing the PCR mix, add the total number of samples andcontrols to be analyzed and find the corresponding volumes of iQ-Design Assay Mix and iQ Multiplex
Powermix in the table.
Total Numberof Samples
and Controls

iQ-Design
Assay, μl

iQ Multiplex
Powermix, μl

Total Numberof Samples
and Controls

1

7.5

12.5

33

iQ-Design
Assay, μl

iQ Multiplex
Powermix, μl

Total Numberof Samples
and Controls

iQ-Design
Assay, μl

iQ Multiplex
Powermix, μl

261

435

65

516

860

2

18

30

34

270

450

66

522

870

3

24

40

35

276

460

67

531

885

4

33

55

36

285

475

68

540

900

5

39

65

37

294

490

69

546

910

6

48

80

38

300

500

70

555

925

7

57

95

39

309

515

71

564

940

8

63

105

40

318

530

72

570

950

9

72

120

41

324

540

73

579

965

10

78

130

42

333

555

74

588

980

11

87

145

43

339

565

75

594

990

12

96

160

44

348

580

76

603

1005

13

102

170

45

357

595

77

612

1020

14

111

185

46

363

605

78

618

1030

15

117

195

47

372

620

79

627

1045

16

126

210

48

381

635

80

636

1060

17

135

225

49

387

645

81

642

1070

18

141

235

50

396

660

82

651

1085

19

150

250

51

405

675

83

657

1095

20

159

265

52

411

685

84

666

1110

21

165

275

53

420

700

85

675

1125

22

174

290

54

429

715

86

681

1135

23

180

300

55

435

725

87

690

1150

24

189

315

56

444

740

88

699

1165

25

198

330

57

453

755

89

705

1175

26

204

340

58

459

765

90

714

1190

27

213

355

59

468

780

91

723

1205

28

222

370

60

477

795

92

729

1215

29

228

380

61

483

805

93

738

1230

30

237

395

62

492

820

94

747

1245

31

246

410

63

498

830

95

753

1255

420

64

845

96

762

1270

32

10 | iQ-Design Assays

252

507

BIO-RAD is a trademark of Bio-Rad Laboratories, Inc. iQ-Check is a trademark of Bio-Rad Europe GmbH in certain jurisdictions. All
trademarks used herein are the property of their respective owner.
This product is intended for internal research use only. The purchaser is responsible for obtaining additional rights, including all
intellectual property rights that may be required.

10000142603 Ver B US/EG