Diagenode Enteric Viral Panel Real-Time PCR kit

DIAGENODE Enteric Viral Panel Real Time PCR kit INSTRUCTIONS FOR USE
Diagenode DBDG-10-4-L096 Version 1
BD 442987 Date of issue: 28.03.2013
Diagenode Enteric Viral Panel
Real-Time PCR kit
Instructions for use
Distributed by
FOR USE WITH THE BD MAX TM SYSTEM

Diagenode sa / CHU - Tour GIGA - B34 – 3 rd Floor // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50
// Mail: info@diagenode.com
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DIAGENODE Enteric Viral Panel Real Time PCR kit INSTRUCTIONS FOR USE
Contents
1. INTRODUCTION ............................................................................................................................... 3
2. GENERAL INFORMATION................................................................................................................. 4
2.1 INTENDED USE ................................................................................................................................... 4
2.2 PATHOGEN INFORMATION ................................................................................................................... 4
2.3 CONTENTS OF KIT .............................................................................................................................. 4
3. MATERIAL ....................................................................................................................................... 5
3.1 MATERIAL REQUIRED (NOT PROVIDED) ................................................................................................. 5
3.2 EQUIPMENT AND MATERIAL IN COMBINATION WITH DIAGENODE ENTERIC VIRAL PANEL REAL-TIME PCR KIT (NOT
PROVIDED) ......................................................................................................................................... 5
4. VALIDATION .................................................................................................................................... 5
5. QUALITY CONTROL .......................................................................................................................... 6
6. REAGENTS STORAGE, HANDLING AND STABILITY .......................................................................... 6
7. SAMPLES COLLECTION, STORAGE AND TRANSPORT ...................................................................... 7
7.1 SAMPLE COLLECTION TYPE ................................................................................................................. 7
7.2 SAMPLE STORAGE .............................................................................................................................. 7
7.3 SAMPLE TRANSPORT .......................................................................................................................... 7
8. WARNINGS AND PRECAUTIONS ...................................................................................................... 8
9. PROTOCOL ...................................................................................................................................... 9
9.1 SAMPLE AND CONTROL PREPARATION .................................................................................................. 9
9.2 RT-PCR MIX PREPARATION ............................................................................................................... 9
9.3 BD MAX TM SYSTEM AMPLIFICATION ................................................................................................... 10
10. RESULTS ..................................................................................................................................... 13
11. TROUBLESHOOTING GUIDE ......................................................................................................... 14
12. PERFORMANCE CHARACTERISTICS ............................................................................................ 15
12.1 ANALYTICAL SENSITIVITY ................................................................................................................. 15
12.2 PRECISION .................................................................................................................................... 15
12.3 ANALYTICAL SPECIFICITY ................................................................................................................. 16
12.4 CLINICAL PERFORMANCE ................................................................................................................ 17
13. TEST LIMITATIONS ...................................................................................................................... 18
14. QUALITY CERTIFICATION ............................................................................................................. 18
15. REFERENCES .............................................................................................................................. 19
16. EXPLANATION OF SYMBOLS ........................................................................................................ 20
17. NOTICE TO PURCHASER .............................................................................................................. 21

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1. Introduction
Real-time polymerase chain reaction, also called quantitative real-time polymerase chain reaction
(qPCR), is a nucleic acid technology used to detect and quantify specific DNA sequences.
This quantification can be measured in absolute or relative number of copies.
Real-time PCR technology allows rapid, specific, and quantitative measurements of the presence of
genes involved in infectious diseases, cancer, and genetic abnormalities.
This powerful technology is used in Diagenode's kits for accurate pathogen detection for a number of
diseases.
One of the prominent attributes of qPCR technology is that amplified DNA is quantified as it accumulates
in the reaction in "real-time" for each PCR amplification cycle, allowing for far more accurate
measurements than with traditional end-point PCR. These real-time measurements are made possible
by quantifying signals from fluorescent dyes that intercalate with the double-stranded DNA produced in
each amplification cycle. Alternatively, one can use labeled DNA oligonucleotide probes with a dye (e.g. a
TaqMan® probe) that subsequently hybridize with the complementary DNA to allow real-time
measurements during qPCR. The resulting fluorescent signals can be closely monitored and quantified
using an instrument such as a thermal cycler adapted for qPCR.
Figure 1: Technology using dual-labeled hydrolysis probes
Figure 2: Specific hybridization of primers and probes to the target gene.
Figure 3: Elongation and release of reporter dye by Taq polymerase.
Diagenode sa / CHU - Tour GIGA - B34 – 3 rd Floor // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50
// Mail: info@diagenode.com

DIAGENODE Enteric Viral Panel Real Time PCR kit INSTRUCTIONS FOR USE
2. General Information
2.1 Intended use
The Diagenode Enteric Viral Panel Real-Time PCR kit as implemented on the BD MAX System is an
automated in vitro diagnostic test for the qualitative detection of Norovirus (genogroups I and II) and
Rotavirus species in stool specimens obtained from patients with signs and symptoms of gastroenteritis.
The detection of Norovirus and Rotavirus nucleic acid from symptomatic patient aids in the diagnosis of
suspected gastro-enteric infections. This test is not intended to differentiate the Norovirus genogroups.
Viral nucleic acid is extracted from the specimen, which undergoes reverse transcription to generate
complementary DNA (cDNA). The target cDNA is amplified using qPCR and then analyzed to detect the
presence or absence of each virus in the panel.
2.2 Pathogen information
Rotavirus is the most common cause of severe diarrhea among infants and young children (1), and is one
of several viruses that cause infections often called stomach flu, despite having no relation to influenza.
It is a genus of double-stranded RNA virus in the family Reoviridae. By the age of five, nearly every child
in the world has been infected with rotavirus at least once (2). However, with each infection, immunity
develops, subsequent infections are less severe (3), and adults are rarely affected (4). There are five
species of this virus, referred to as A, B, C, D, and E. Rotavirus A, the most common, causes more than
90% of infections in humans.
Norovirus is an RNA virus that causes approximately 90% of epidemic non-bacterial outbreaks of
gastroenteritis around the world (5), and may be responsible for 50% of all foodborne outbreaks of
gastroenteritis in the US (6). Norovirus I and II are the both genogroups the most important in human and
affects people of all ages. The viruses are transmitted by faecally contaminated food or water, by
person-to-person contact (7) and via aerosolization of the virus and subsequent contamination of surfaces
(8).
2.3 Contents of kit
This kit is used with the BD MAX TM System.
Diagenode Enteric Viral Panel Real-Time PCR kit: 96 PCR reactions (12,5 µL PCR volume), one box :
I. Enteric Viral Panel and internal control RNA double-dye probe & primers (4 tubes):
Enteric Viral Panel double-dye probe & primers with Noro I (FAM, 520 nm), Noro II (YD, 549 nm),
Rota (TR, 603 nm), and RNA extraction & inhibition control double-dye probe & primers (Cy5, 662
nm): 65 µL each, red tubes.
II. Enteric Viral Panel positive control: 120 µL, green tube.
III. Enteric Viral Panel negative control (H2O PCR grade): 120 µL, sky blue tube.
IV. H2O PCR grade: 500 µL, sky blue tube.
V. Optima PLUS Master Mix: 720 µL, white tube
VI. RNA virus culture inactivated (internal control): 1000 µL, yellow tube

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3. Material
3.1 Material required (Not provided)
• Phosphate buffered saline (PBS)
• Microcentrifuge
• Pipettes (accurate between 1-1000 µL)
• Sterile pipette tips with filters
• Powder-free gloves
• Vortex mixer
• Lab coat
• RNase/DNase-free microcentrifuge tubes (1.5 and/or 2 mL)
• RNase/DNase free water
• Laminar flow hood
• A sterile and crew-cap container for the collection of stool samples
• Stopwatch or timer
• Precision balance
3.2 Equipment and material in combination with Diagenode Enteric Viral Panel Real-Time
PCR kit (Not provided)
• Real-time PCR instrument: BD MAX System (Ref: 441916)
• BD MAX RNA extraction kit RNA-3 (Ref : 437522): RNA Extraction Reagent (E2-R)
RNA Sample Preparation Reagent (SP2-R)
DNase Reagent (D1)
RNA Unitized Reagent Strips
• BD MAX PCR Cartridges (Ref : 437519)
4. Validation
Diagenode Enteric Viral Panel Real-Time PCR kit has been validated on the parameters as described
below:
Collected samples
PCR platform
Stools
BD MAX TM System
Diagenode sa / CHU - Tour GIGA - B34 – 3 rd Floor // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50
// Mail: info@diagenode.com

DIAGENODE Enteric Viral Panel Real Time PCR kit INSTRUCTIONS FOR USE
5. Quality Control
• Quality Control requirements must be performed in conformance with local, state and/or federal
regulations or accreditation requirements and your laboratory’s standard quality control procedures.
• Quality control procedures are intended to monitor reagent and assay performance.
Control type
Positive
Negative
Internal
For monitoring the following reagents and assay performance
Substantial reagent failure including primer and probe integrity
Reagent and/or environmental contamination
Failure in lysis and extraction procedure
PCR inhibition in individual samples and reagent failure or process error
• Test all positive control and the internal control prior to running samples with each new kit lot to
ensure all reagents and kit components are working properly.
• Good laboratory practice recommends including the internal control in each nucleic acid isolation
run. The internal control should be treated as a sample.
• Never run the positive control through nucleic acid isolation.
• Inclusion of one negative control and at least one positive control is recommended in each
amplification / detection run performed.
• Failure of controls invalidates the run and results should not be reported.
- If the positive control is not positive within the specified Ct range but the negative control is valid,
repeat testing should be done starting from the original sample and using new aliquot of the
positive control. If repeat results are still invalid, results should not be reported and testing
should be repeated from a new sample should be collected and tested.
- If the internal control is not positive within the specified Ct range or the negative control is invalid,
repeat testing should be done starting from the original sample using a new internal control and a
new negative control. If repeat results are still invalid, results should not be reported and a new
sample should be collected and tested (see point 11. Troubleshooting Guide).
6. Reagents Storage, Handling and Stability
Store all reagents (opened and unopened) at ≤ -20°C until the expiry date as defined below:
Condition Storage temperature Stability
Unopened reagent ≤ -20°C Expiry date stated on the label
Always check the expiry date on the reagent labels.
We recommend not exposing to more than 6 freeze-thaw cycles to avoid the degradation of compounds.
Diagenode Enteric Viral Panel Real-Time PCR kit is shipped frozen, should arrive frozen and should be
stored frozen at ≤-20°C after receipt. If the kit contents are not frozen, contact your local BD
representative.
Protect reagents from light.
Caution
Storage of the kit at room temperature can lead to the degradation of the kits components. This will lead
to a decreased sensitivity and we would strongly advise using a new kit.

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7. Samples Collection, Storage and Transport
7.1 Sample Collection Type
The PCR test is performed using liquid or soft stool samples.
Standard collection procedures are appropriate to obtain raw stool. Raw stool should be placed in a
sterile and screw-cap container that can be adequately sealed.
7.2 Sample Storage
Stool samples must be stored at 2 to 8°C for up to 24 hours or frozen at -20°C or -70°C for storage
longer than 24 hours.
Stool samples should be stored in sterile and screw-cap container.
The sensitivity of the PCR could be reduced through repeated freezing and thawing of stool samples;
therefore, avoid freeze/thaw cycles.
7.3 Sample Transport
Stool samples must be transported in dry sterile container.
The samples must be transported following the local and national regulations for the transport of
etiologic agent.
In order to avoid degradation of nucleic acid, we advise shipping at -20°C if transport time is more than
24 hours.
In the laboratory, samples may be transported at room temperature.
Diagenode sa / CHU - Tour GIGA - B34 – 3 rd Floor // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50
// Mail: info@diagenode.com

DIAGENODE Enteric Viral Panel Real Time PCR kit INSTRUCTIONS FOR USE
8. Warnings and Precautions
If you receive a damaged parcel or thawed kits, please contact our local BD
representative. Do not use the kit.
General information
• Read all instruction before performing the experiment.
• Use of this product should be limited to personnel who have been trained in the techniques of Real-
Time PCR.
• For in vitro diagnostic use.
• This assay is for use with liquid or soft raw stool specimens.
Precautions
• Do not use the kit if the label that seals the outer box is broken.
• Do not use reagents if the protective box is open or torn upon arrival.
• Do not use reagents if the tube has been opened or damaged.
• Do not mix reagents from different kits and/or lots.
• Do not use expired reagents and/or materials.
• Do not mix caps between tubes or re-use caps as contamination may occur and compromise
test results.
• Aerosol resistant tips must be used for all PCR mixtures.
• Performing the assay outside of the recommended time ranges may produce invalid
results. Assays not performed within specified time ranges should be repeated.
• The PCR laboratory (safety cabinet, DNA/ RNA extraction platform, bench coat, etc.) must be cleaned
after each PCR experiment with appropriate solutions.
• Additional controls may be tested according to guidelines or requirements of local,
state, provincial and/or federal regulations or accrediting organizations.
• In cases where other PCR tests are conducted in the same general area of the laboratory, care must
be taken to ensure that the Diagenode Enteric Viral panel Real Time PCR kit, BD MAX RNA
Extraction kit RNA-3, any additional reagents required for testing, and the BD MAX System are not
contaminated. Gloves must be changed before manipulating reagents and cartridges.
• Discard the kit with a suspected contamination.
• Always handle specimens as if they are infectious and in accordance with safe
laboratory procedure such as those described in local legislation documentation.
• Wear protective clothing and disposable gloves while handling kit reagents. Wash and disinfect hands
thoroughly after performing the test.
• The kit should not be used by somebody displaying symptoms of the disease being detected by
the kit.
• Do not pipette by mouth.
• Do not ingest any components from the kit.
• Do not smoke, drink, or eat in areas where specimens or kit reagents are being handled.
• The experiments should not be carried out during pregnancy due to risks to the newborn baby.
• Dispose of unused reagents and waste in accordance with country, federal, provincial, state and local
regulations.

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9. Protocol
Collect specimen and label appropriately (see point 7.1 Samples Collection Type)
9.1 Sample and control preparation
9.1.1 Sample pretreatement
• Weigh 100 mg of stool samples with a precision balance.
• Add 1 mL PBS to 100 mg of stool.
• Vortex for 1 minute.
• Allow mixture to rest for 10 minutes at room temperature.
• Vortex for another 1 minute.
• Centrifuge at 1500 rpm for 15 seconds.
• Dilute the supernatant 1/10 in PBS.
• Pipette 500 µL of the supernatant and pipette 10 µL of RNA internal control ( yellow tube)
into one Sample Preparation Reagent tube.
• Close each Sample Preparation Reagent tube and vortex for 10 seconds.
9.1.2 Diagenode negative and positive control preparation (optional)
To validate the run and for result interpretation, you need to perform a positive and negative control:
• For the positive control:
Pipette 10 µL of RNA internal control (yellow tube) and 500 µL of RNase/DNase free water into
one Sample Preparation Reagent tube. Re-cap the tube, vortex, and un-cap the tube prior to use.
• For the negative control:
Pipette 10 µL of Diagenode negative control (sky blue tube), 10 µL of RNA internal control (yellow
tube) and 490 µL of RNase/DNase free water into one Sample Preparation Reagent tube. Re-cap
the tube, vortex, and un-cap the tube prior to use.
9.2 RT-PCR Mix preparation
1. Count the total number of samples and controls to be tested in one run.
2. Shortly before running the assay, prepare the following complete master mix by multiplying each
component volume by the total number of samples, plus 20% overage accounting for pipetting errors
(see table below).
Optima PLUS Master Mix (white tube) ……………………………….6.75 µL
Enteric Viral panel primers and probes (red tube) ………………2.5 µL
H2O PCR grade (sky blue tube) ….………………………………………3.25 µL
Final volume for one strip: ………………………………..……… 12.5 µL
Diagenode sa / CHU - Tour GIGA - B34 – 3 rd Floor // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50
// Mail: info@diagenode.com

DIAGENODE Enteric Viral Panel Real Time PCR kit INSTRUCTIONS FOR USE
Example of calculation for several strips/reactions:
Number of
samples
Percentage
error of
pipetting
Primers/Probe
(µL)
Optima PLUS
Master Mix
(µL)
H 2 O
(PCR grade)
(µL)
Total
(µL)
3 20% 9 24.3 11.7 45.0
4 20% 12 32.4 15.6 60.0
5 20% 15 40.5 19.5 75.0
6 20% 18 48.6 23.4 90.0
7 20% 21 56.7 27.3 105.0
8 20% 24 64.8 31.2 120.0
9 20% 27 72.9 35.1 135.0
10 20% 30 81.0 39.0 150.0
11 20% 33 89.1 42.9 165.0
12 20% 36 97.2 46.8 180.0
13 20% 39 105.3 50.7 195.0
14 20% 42 113.4 54.7 210.1
15 20% 45 121.5 58.6 225.1
16 20% 48 129.6 62.5 240.1
17 20% 51 137.7 66.4 255.1
18 20% 54 145.8 70.3 270.1
19 20% 57 153.9 74.2 285.1
20 20% 60 162.0 78.1 300.1
21 20% 63 170.1 82.0 315.1
22 20% 66 178.2 85.9 330.1
23 20% 69 186.3 89.8 345.1
24 20% 72 194.4 93.7 360.1
9.3 BD MAX TM System amplification
Note: Refer to the BD MAX System User’s Manual for detailed instructions.
9.3.1 Creating PCR test for Diagenode EVP (Enteric Viral Panel)
Note: If you already have created the Diagenode EVP test, you can skip step 9.3.1 and go directly to
9.3.2.
1. On the « Run » screen of the BD MAX TM System, select the « Test Editor » tab
2. Click the « Create Test » button.
3. In the « Test Name » window, name your test: Diagenode EVP.
4. In the « Extraction Type » drop down menu, select RNA-3.
5. In the « Master Mix Format » drop down menu, choose« Research MM, Probes/Primers Add
Manually ».

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6. In « Channel Settings », set « Gains » and « Threshold » as follow :
Channel
475/520
530/565
585/630
630/665
680/715
7. In « GuardRail », select « Default »
8. In « Test Details », enter the PCR profile :
Gain
40
60
60
60
0
Threshold
150
250
300
150
/
New Step
Cycle
1
Type
Profile type
Time (s)
1800.0
Hold
Temp (°C)
50.3
Detect
New Step
Cycle
1
Type
Profile type
Time (s)
600.0
Hold
Temp (°C)
95.0
Detect
New Step
Cycle
45
Type
Profile type
Time (s)
30.1
30.0
30.0
3-Temperature
Temp (°C)
95.0
55.0
68.0
Detect
9. Click the « Spectral Cross Talk » tab and enter the following parameters:
Excitation
Channel
False Receiving Channel
475/520 530/565 585/630 630/665 680/715
475/520 2.1 0.0 0.0 0.0
530/565 1.2 0.0 0.0 0.0
585/630 0.0 0.3 2.7 0.0
630/665 0.0 0.0 4.9 0.0
680/715 0.0 0.0 0.0 4.7
10. Click the « Save Test » button.
Diagenode sa / CHU - Tour GIGA - B34 – 3 rd Floor // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50
// Mail: info@diagenode.com

DIAGENODE Enteric Viral Panel Real Time PCR kit INSTRUCTIONS FOR USE
9.3.2 BD MAX TM Rack set up
Please refer to BD MAX User’s Manual for additional information.
1. Load the BD MAX racks with the appropriate number of RNA Unitized Reagent Strips (URS).
Gently tap each URS onto a hard surface to ensure that the liquids are at the bottom.
2. Snap the RNA Extraction Reagent tube (white foil) and the DNase Reagent tube (yellow foil) into their
corresponding positions in the URS (see figure 1).
3. Pipette 12.5 μL of Complete Master Mix (prepared in section 9.2) into the snap-in Ready RNA tube
(position 3) of the RNA URS. Make sure there are no bubbles and the liquid is at the bottom of the
tube.
4. For the positive control sample URS, pipette 1 μl of positive control (green tube) into the Ready RNA
tube (position 3) in addition to the Master Mix.
9.3.3 BD MAX TM Instrument set up
1. Select the tab on the screen of the BD MAX System.
2. In the “Assay” drop down menu, select Diagenode EPV (if not already created see Section 9.3.1).
3. Enter the Sample Preparation Reagent tube barcode corresponding to each specimen/patient or
control using the barcode scanner or manual entry. Start with Position 1 of Rack A.
4. Place the opened Sample Preparation Reagent tube into its corresponding position in the BD MAX TM
Rack(s).
5. Enter the specimen/patient identification information into the work list, using either the barcode
scanner or manual entry. Continue until all Sample Preparation Reagent tubes are entered. Ensure
that the specimen/patient ID and the Sample Preparation Reagent tubes are accurately matched.
6. Load the rack(s) into the BD MAX TM System (Rack A is positioned on the left side of the BD MAX
System and Rack B on the right side).
7. Load the BD MAX TM PCR cartridge(s).
8. Close the BD MAX System door.
9. Click “Start Run”

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10. Results
1. In main menu, click the “Results” button.
2. Double click on your run in the list and click on the new tab “Run …..”
3. In the “Print” tab, select: Result
Protocol Details
PCR
4. Click on “Create Report” and then on click the “Print Report” button.
Results interpretation with BD MAX TM System software 2.7.4 or 2.7.0
1. For a run to be valid (optional):
a. No BD MAX TM System failures.
b. Negative control has CT value of -1 for all channels except 630/665.
c. Positive control has CT value of 30 ±3.3 for channels 475/520, 530/565, and 585/630.
2. If valid, interpret the specimen results following the chart below:
a. A CT value of 0 indicates that there was no CT value calculated. That sample’s curve must be
reviewed manually with threshold line adjustment to generate a recalculated CT value.
b. A CT value of -1 indicates that no amplification occurred.
c. Any other CT value must be interpreted in correlation with PCR curves (“PCR Analysis” tab) and
according to the following table:
CT 475/520
Norovirus I
CT 530/565
Norovirus II
CT 585/630
Rotavirus
CT 630/665
Internal Control
Results Interpretation
-1 -1 -1 ≤ 36
Norovirus (I/II) and Rotavirus
RNA Not Detected.
ANY -1 -1 ANY Norovirus (I/II) RNA detected.
Rotavirus RNA Not Detected.
-1 ANY -1 ANY
Norovirus (I/II) RNA detected.
Rotavirus RNA Not Detected.
-1 -1 ANY ANY
Rotavirus RNA detected.
Norovirus (I/II) RNA Not Detected.
ANY ANY -1 ANY
Norovirus (I/II) RNA detected.
Rotavirus RNA Not Detected.
ANY -1 ANY ANY
Norovirus (I/II) and Rotavirus RNA
detected.
-1 ANY ANY ANY
Norovirus (I/II) and Rotavirus RNA
detected.
ANY ANY ANY ANY
Norovirus (I/II) and Rotavirus
RNA detected.
-1 -1 -1
> 36 Inhibitory Specimen*.
Or -1
No diagnosis can be established
from this test.
*For Inhibitory specimens: Repeat test with the original specimen stored at 2-8°C by preparing a new
Sample Preparation Reagent tube from diluted (with PBS) or undiluted specimen. Alternatively, test a
newly collected specimen.
Diagenode sa / CHU - Tour GIGA - B34 – 3 rd Floor // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50
// Mail: info@diagenode.com

DIAGENODE Enteric Viral Panel Real Time PCR kit INSTRUCTIONS FOR USE
11. Troubleshooting guide
Refer to “the troubleshooting” section of the BD MAX TM System User’s Manual for additional information.
Description Possible Causes Corrective Actions
PCR inhibited by
exogenous or
endogenous
substances
Repeat sample testing.
Target and internal control do not
amplify.
Target amplifies, but the internal control
does not amplify.
Spike in all amplification curves at same
point.
Very low fluorescence across all
samples, including internal control
Liquid handling
problem
Target out competed
internal control for
resources during
reaction
Power surge
Primer or probe
degradation
Instrument issue
Check URS and Microfluidic
Cartridge to determine where
liquid handling problem
occurred and re-run sample. If
problem persists, contact your
local BD representative
Verify sample has early
amplification.
Verify that system is connected
to the UPS (Uninterruptible Power
Supply).
Check kit expiration date and
verify kit was stored correctly.
Refer to BD MAX User’s
Manual.
For other trouble shootings/questions, please contact your local BD representative or the
Diagenode customer support.

Page | 15
12. Performance Characteristics
12.1 Analytical sensitivity
The analytical detection limit in consideration of the purification (LOD) was assessed for Norovirus I and
II in stool matrix. The analytical detection limit in consideration of the purification is determined using
quantified inactivated Norovirus I and II pathogens. No quantified genomic standard for Rotavirus was
available at the time the analytical sensitivity test was performed. Therefore the LOD for the Rotavirus
has been performed with plasmid DNA directly spiked in the PCR Master Mix solution. The number of
copies/reaction was then converted in theoretical copies/ml of sample assuming an extraction efficiency
of 100%.
To determine the analytical sensitivity in consideration of the purification of the Diagenode Enteric Viral
Panel Real-Time PCR Kit for the detection of Norovirus (I/II) in stool, Norovirus I and Norovirus II dilution
series have been set up in pools of negative stools.
To determine the analytical sensitivity of the Diagenode Enteric Viral Panel Real-Time PCR Kit for the
detection of Rotavirus, dilution series of a specific plasmid containing the Rotavirus target sequence
have been set up directly in the PCR Master Mix solution.
Samples were analysed on the BD MAX TM Instrument in combination with the Diagenode Enteric Viral
Panel Real-Time PCR Kit. For Rotavirus, the extraction step was bypassed. Testing was carried out on
different days on 24 replicates. The results were determined by a probit analysis. Analytical sensitivity
(LOD) as defined as the lowest concentration at which ≥ 95% of replicates are tested positive are
presented in the table below.
LOD 95%
Copies/PCR
Copies/mL
Norovirus I NA 5 151 (312 to 10 000)*
Norovirus II NA 587
Rotavirus 30 397
* The sensitivity of Norovirus I is strongly influenced by the stool specimen.
In the present sensitivity study the LOD ranged from 312 to 10 000 copies/mL
depending of the pool of stool used
12.2 Precision
The precision data of the Diagenode Enteric Viral Panel Real-Time PCR Kit have been collected by
means of the BD MAX TM Instrument and allow the determination of the intra-run variability (variability of
multiple results of samples of the same concentration within one experiment) and the inter-run
variability (variability of multiple results of the assay generated by four different runs carried out on
different days) of Norovirus I/II and Rotavirus detection. The data obtained were used to determine the
standard deviation and the coefficient of variation for the pathogens detected.
Precision data for Norovirus I and II have been collected using stool matrix spiked with the
correspondent inactivated pathogens at high and low (closed to the respective LODs) concentrations of
the pathogens. No quantified genomic standard for Rotavirus was available at the time the precision
tests were performed. Therefore precision data for Rotavirus has been collected using plasmid DNA
directly spiked in the PCR Master Mix solution at high and low (closed to the LOD) concentrations of
Rotavirus.
Testing was performed by different operators on different days. The precision data were calculated on
basis of the Ct values of the amplification curves as following: intra-assay variation = mean CV of each
Diagenode sa / CHU - Tour GIGA - B34 – 3 rd Floor // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50
// Mail: info@diagenode.com

DIAGENODE Enteric Viral Panel Real Time PCR kit INSTRUCTIONS FOR USE
individual run; inter-assay variation = CV of the mean Ct value of each individual run. Results are
presented in the table below.
Test
Intra-assay Noro I
Inter-assay Noro I
Intra-assay Noro II
Inter-assay Noro II
Intra-assay Rota
Inter-assay Rota
Matrix
Pathogen
Standard Coefficient of
Mean Ct
concentration
deviation 1 variation (%) 2
Stool 312 c/mL 34.6 1.26 3.54
Stool 10 000 c/mL 32.5 0.53 0.85
Stool 312 c/mL 34.6 1.25 3.62
Stool 10 000 c/mL 32.5 2.35 0.67
Stool 2500 c/mL 30.6 0.45 1.46
Stool 625 c/mL 31.5 0.35 1.12
Stool 2500 c/mL 30.6 0.55 1.81
Stool 625 c/mL 31.5 0.43 1.38
NA 532 c/mL 33.3 0.50 1.51
NA 2128 c/mL 31.8 0.25 0.78
NA 532 c/mL 33.3 0.50 1.32
NA 2128 c/mL 31.8 0.31 0.97
1
Values correspond to the mean SD of each individual runs for intra-run variation and to the SD of the
mean Ct value of each individual run for the inter run variation
2
Values correspond to the mean CV of each individual runs for intra-run variation and to the CV of the
mean Ct value of each individual run for the inter run variation
12.3 Analytical specificity
The specificity of the Diagenode Enteric Viral Panel Real-Time PCR Kit is first and foremost ensured by
the selection of the primers and probes, as well as the selection of stringent reaction conditions. The
primers and probes were checked for possible homologies to all in gene banks published sequences by
sequence comparison analysis.
To determine the experimental specificity of the Diagenode Enteric Viral Panel Real-Time PCR Kit, the
pathogens listed in the following table have been tested for cross-reactivity. None of the tested
pathogens has been reactive.
Pathogens
Bacillus cereus
Campylobacter jejuni
Clostridium difficile
Clostridium perfringens
Cryptosporidium hominis
Cryptosporidium parvum
Entamoeba dispar
Entamoeba histolytica
Enterocytozoon bieneusi
Enterovirus 71
Giardia intestinalis
Human Coxsavirus B4
Human Echovirus 13
Parechovirus 1
Salmonella enterica
Shigella flexneri
Staphylococcus aureus
Yersinia enterocolitica

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12.4 Clinical performance
Performance characteristics of the Diagenode Enteric Viral Panel Real-Time PCR kit were established
during a retrospective study at one reference laboratory in comparison to a validated PCR reference
method.
A total of 225 samples (100 negative samples, 25 Norovirus I positive samples, 50 Norovirus-II positive
samples and 50 Rotavirus positive samples) were retrospectively tested with the Diagenode Enteric Viral
Panel Real-Time PCR kit on the BD MAX TM . Samples consisted of 100 mg stool suspended in 0.4 mL
phosphate-buffered saline containing 2% polyvinylpolypyrolidone for the reference method and in 1mL
phosphate-buffered saline for the Diagenode Enteric Viral Panel Real-Time PCR kit. 200 µL of sample
supernatant were initially extracted on the MagNa-Pure LC, Roche using the TNAI High Performance Kit
(50 µL eluate) and analysed with the Multiplex Real-time PCR reference method (9) in combination with
the BioRad CFX96 real-time system, the Biorad 5x iScript RT and the Supermix Qiagen 2x HotStar
Mastermix. 500 µL of sample supernatant 1/10 prediluted in PBS were loaded on the BD
MAX TM cartridge. Height invalid samples (no IC detection and no target-specific detection) and one
rotavirus overloaded samples (the software is unable to calculate a Ct value because the viral load is too
high) were excluded from the statistical analysis.
Diagnostic sensitivity, diagnostic specificity and accuracy were calculated for rotavirus and Norovirus
(I/II). Results are summarized in the following tables:
Rotavirus
Enteric Viral Panel kit
on BD MAX TM
Positive Negative
Norovirus
Enteric Viral Panel kit on
BD MAX TM
Positive
(I/II)
Negative
(I/II)
Reference method
Positive
Negative
49 0
7 160
Reference method
Positive
(I/II)
Negative
(I/II)
64
(21/43)
5
(4/1)
6
(0/6)
142
(N/A)
Diagnostic sensitivity (lower Bound 95%CI):
100% (93%) 91% (83%)
Diagnostic specificity (lower Bound 95%CI):
Accuracy:
96% (92%) 97% (92%)
Κ = 0.91
(almost perfect agreement)
Κ = 0.88
(almost perfect agreement)
Diagenode sa / CHU - Tour GIGA - B34 – 3 rd Floor // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50
// Mail: info@diagenode.com

DIAGENODE Enteric Viral Panel Real Time PCR kit INSTRUCTIONS FOR USE
13. Test limitations
• All reagents must exclusively be used for in vitro diagnostics.
• A strict compliance with the user manual is required for optimal results.
• This test has been validated for use with liquid or soft stool samples.
• This test has been validated for use on the BD MAX TM System version 2.7.0 or 2.7.4.
• This test has been validated for the detection of Norovirus and Rotavirus and does not allow
differentiation of Norovirus genogroups.
• Negative results do not preclude infection and should not be used as the sole basis for diagnosis,
treatment or other management decisions.
• The detection of viral nucleic acid is dependent upon proper specimen collection, handling,
transportation, storage and preparation. Failure to observe proper procedures in any one of these
steps can lead to incorrect results. There is a risk of false negative values resulting from improperly
collected, transported or handled samples.
• There is a risk of false negative values due to the presence of sequence variants in the viral targets of
the assay, procedural errors, amplification inhibitors in samples, or inadequate number of organisms
for amplification.
• False negative results may occur due to loss of nucleic acid. The inhibition control has been added to
the test to aid in the identification of samples that contain inhibitors to PCR amplification. The control
does not indicate whether or not nucleic acid has been lost due to inadequate collection, transport or
storage of samples.
• There is a risk of false positive values resulting from cross-contamination by target organism, their
nucleic acids or amplified product, or form non-specific signal in the assay.
14. Quality Certification
Diagenode has obtained ISO 9001 and ISO 13485 certifications for design, manufacture and sale of IVD
devices using Nucleic Acid technology for infectious diseases.
Diagenode Enteric Viral Panel Real-Time PCR kit is tested against predetermined specifications to
ensure the product quality.

Page | 19
15. References
(1) Dennehy PH (2000). "Transmission of rotavirus and other enteric pathogens in the home". Pediatr.
Infect. Dis. J. 19 (10 Suppl): S103–5.
(2) Velázquez FR, Matson DO, Calva JJ, Guerrero L, Morrow AL, Carter-Campbell S, Glass RI, Estes MK,
Pickering LK, Ruiz-Palacios GM (1996). "Rotavirus infections in infants as protection against subsequent
infections". N. Engl. J. Med. 335 (14): 1022–8.
(3) Linhares AC, Gabbay YB, Mascarenhas JD, Freitas RB, Flewett TH, Beards GM (1988). "Epidemiology
of rotavirus subgroups and serotypes in Belem, Brazil: a three-year study". Ann. Inst. Pasteur Virol. 139
(1): 89–99.
(4) Bishop RF (1996). "Natural history of human rotavirus infection". Arch. Virol. Suppl. 12: 119–28.
(5) Lindesmith L, Moe C, Marionneau S, et al. (2003). "Human susceptibility and resistance to Norwalk
virus infection". Nat. Med. 9 (5): 548–53.
(6) Widdowson MA, Sulka A, Bulens SN, et al. (2005). "Norovirus and foodborne disease, United States,
1991-2000". Emerging Infect. Dis. 11 (1): 95–102.
(7) Goodgame R (2006). "Norovirus gastroenteritis". Curr Gastroenterol Rep 8 (5): 401–8.
(8) Said MA, Perl TM, Sears CL (November 2008). "Healthcare epidemiology: gastrointestinal flu:
norovirus in health care and long-term care facilities". Clinical Infectious Diseases : an Official
Publication of the Infectious Diseases Society of America 47 (9): 1202–8.
(9) Diagnosis of viral gastroenteritis by simultaneous detection of Adenovirus group F, Astrovirus,
Rotavirus group A, Norovirus genogroups I and II, and Sapovirus in two internally controlled multiplex
real-time PCR assays. Noortje M. van Maarseveen1, Els Wessels, Caroline S. de Brouwer, Ann C.T.M.
Vossen, Eric C.J. Claas. Journal of Clinical Virology 49 (2010) 205–210
Noortje M. van Maarseveen, Els Wessels, Caroline S. de Brouwer, Ann C.T.M. Vossen, Eric C.J. Claas
“Diagnosis of viral gastroenteritis by simultaneous detection of Adenovirus group F, Astrovirus,
Rotavirus group A, Norovirus genogroups I and II, and Sapovirus in two internally controlled multiplex
real-time PCR assays”. Journal of Clinical Virology 49 (2010) 205–210
S.-Q. Zeng, A. Halkosalo, M. Salminen, E.D. Szakal, L. Puustinen, T. Vesikari.
“One-step quantitative RT-PCR for the detection of rotavirus in acute gastroenteritis”.
Journal of Virological Methods 153 (2008) 238–240
Young Bin Park, You-Hee Cho, GwangPyo Ko
“A duplex real-time RT-PCR assay for the simultaneous genogroup-specific detection of noroviruses in
both clinical and environmental specimens”. Virus Genes (2011) 43:192–200
Diagenode sa / CHU - Tour GIGA - B34 – 3 rd Floor // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50
// Mail: info@diagenode.com

DIAGENODE Enteric Viral Panel Real Time PCR kit INSTRUCTIONS FOR USE
16. Explanation of symbols
Catalog number
Batch Code
Contains sufficient for tests
Upper limit of temperature
Use by yyyy-mm
In vitro diagnostic medical device
Consult instructions for use
Biological risks
Control
Negative control
Positive control
Keep away from sunlight
Manufacturer

Page | 21
17. Notice to Purchaser
This product is optimized for use in the polymerase chain reaction (PCR) covered by patents owned by Roche
Molecular Systems, Inc., and F. Hoffmann-La Roche ltd. (Roche). No license under these patents to use the
PCR process is conveyed expressly or by implication to the purchaser by the purchase of this product.
A license to use the PCR process for certain research and development activities accompanies the
purchase of certain reagents from licensed suppliers, when used in conjunction with an Authorized
Thermal Cycler, or is available from Applied Biosystems. Further information on purchasing licenses to
practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems,
850 Lincoln Center Drive, Foster City, California 94404 or at Roche Molecular Systems, Inc, 1145 Atlantic
Avenue, Alameda, California 94501.
BD MAX TM system is a trademark of Becton, Dickinson and Company.
Diagenode sa / CHU - Tour GIGA - B34 – 3 rd Floor // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50
// Mail: info@diagenode.com

DIAGENODE Enteric Viral Panel Real Time PCR kit INSTRUCTIONS FOR USE
Diagenode s.a.
Avenue de l’hôpital,1 • CHU - Tour GIGA - B34 -3rd Floor • 4000 Liège (Sart Tilman)• Belgium
Tel: +32 4 364 20 50 • Fax: +32 4 364 20 51 • info@diagenodediagnostics.com

Page | 23
Diagenode sa
CHU, Tour GIGA, B34 3 rd Floor
Avenue de l’hôpital, 1
4000 Liège (Sart Tilman)
BELGIUM
Tel. +32 4 364 20 50
info@diagenodediagnostics.com
www.diagenodediagnostics.com
Diagenode sa / CHU - Tour GIGA - B34 – 3 rd Floor // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50
// Mail: info@diagenode.com