Varicella Zoster Virus - Diagenode Diagnostics

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Ref.: Dia-VZV-050.Vs1
27.08.2014
Varicella Zoster Virus
Real-Time PCR
User Manual
Available on:
ABI 7000 / 7300 / 7500 / 7900
Roche LightCycler 480 / 2.0
Biorad Icycler / IQ5 / CFX96
Qiagen Rotor-Gene 6000
Stratagene MX3000P / 3500P
Cepheid Smart Cycler
In combination with:
DIA-EIC/DNA(YD)-050
DIA-EIC/DNA(DR)-050
DIA-EIC/DNA(TR)-050
DIA-EIC/DNA(Cy5)-050
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Diagenode sa / LIEGE SCIENCE PARK // Rue Bois Saint-Jean, 3 // 4102 Seraing (Ougrée) // Belgium // Phone: (+32) 4 364 20 50 // Mail: [email protected]
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PAGE
Contents
General information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Pathogen information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Product description. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Warnings and precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Material required, not provided. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Human samples Collection, Storage and Transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1. Samples' Collection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2. Samples' Storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3. Samples' Transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
A. Procedure to be followed
A.1. Extraction procedure (see point II. Sample Extraction/DNA Isolation). . . . . . . . . . . . . . . . . . . 8
A.2. PCR procedure (see point III. PCR Reaction Set-up). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
A.3. A
mplification and real-time system procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
A.4. Interpretation of results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
B. Annexes of the procedure (points I-II-III-IV-V-VI)
I. DNA extraction & inhibition control (optional) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
II. Sample Extraction/DNA Isolation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
III. PCR Reaction Set-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
IV. PCR profile. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
V. Selection of the appropriate dyes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
ABI 7000-7300-7500-7900. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Roche Lc480 - Lc2.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Biorad iCycler-IQ5-CFX96. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Stratagene MX3000P-3005P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Qiagen Rotor-Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Cepheid SmartCycler II. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
VI. Interpretation of results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Troubleshooting guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Specifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Sensitivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Specificity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Diagnostic evaluation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Product Use Limitations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Quality Control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Explanation of symbols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Notice to purchaser. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
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DIAGENODE VARICELLA ZOSTER VIRUS REAL-TIME PCR USER MANUAL
General Information
Diagenode’s Varicella zoster virus Real Time PCR is an in vitro diagnostic assay used by a clinical
laboratory and by trained laboratory scientists.
Diagenode’s Varicella zoster virus Real Time PCR has been validated on:
• ABI (7000 / 7300 / 7500 /7900)
• Roche (Lc2.0 / Lc480)
• Biorad (iCycler / IQ5 / CFX96)
• Qiagen Rotor-Gene (3000 / 6000)
• Stratagene (MX3000P / 3500P)
• Cepheid (SmartCycler II)
Pathogen Information
Varicella zoster virus (VZV) is one of eight herpes viruses known to infect humans. It commonly
causes chicken-pox in children and both shingles and postherpetic neuralgia in adults.
Primary VZV infection results in chickenpox (varicella), which may rarely result in complications
including encephalitis or pneumonia. Even when clinical symptoms of chickenpox have resolved,
VZV remains dormant in the nervous system of the infected person (virus latency), in the trigeminal
and dorsal root ganglia (Steiner et al., 2007). In about 10-20% of cases, VZV reactivates later in
life producing a disease known as herpes zoster or shingles. Serious complications of shingles
include postherpetic neuralgia, zoster multiplex, myelitis, herpes ophthalmicus, or zoster sine
herpete.
Product Description
The product contains primers, probes and a positive control. The master mix is not included.
Varicella zoster virus Real-Time PCR: 50 PCRs (50 μl PCR volume), 2 pockets
I. Primers & probe’s pocket:
1. Varicella zoster virus primers & double-dye probes (FAM, emission 520 nm):
250 μl, mallow tube.
2. ddH2O: 1500 μl, dark blue tube.
II. Positive control’s pocket:
1. Varicella zoster virus positive control: 250 μl, red tube.
The Varicella zoster virus Real-Time PCR (DIA-VZV-050) is used in combination with the DNA
extraction & inhibition control Real-Time PCR (DIA-EIC/DNA-050).
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DNA extraction & PCR inhibition control Real-Time PCR: 50 PCRs (50 μl PCR volume), 1 box.
I. (DNA virus) primers & double-dye probe:
If Yellow Dye (DIA-EIC/DNA(YD)-050), emission 548 nm : 250 µl, green tube.
If Orange Dye (DIA-EIC/DNA(DR)-050), emission 575 nm: 250 μl, blue tube.
If Texas Red (Dia-EIC/DNA(TR)-050), emission 603 nm: 250 μl, red gtube.
If Cy5 (DIA-EIC/DNA(Cy5)-050, emission 662 nm: 250 μl, dark blue tube.
II. DNA virus culture: 500 μl, yellow tube.
Storage
Varicella zoster virus primers, probe and positive control must be stored at - 20°C.
Virus-containing tube from the DNA extraction and PCR inhibition control must be stored at
- 20°C.
Please check production date mentioned on the pockets. The product must be store during 24
months starting the production date. Repeated thawing and freezing must be avoided, make
aliquots. It is also recommended to manipulate the entire kit’s components on ice.
STORE IN DARK PLACE.
Warnings and Precautions
Extraction/PCR
• Diagenode’s real-time PCR kits must be used by scientists/laboratory technicians with
a strong know-how of molecular biology and more particularly with real-time PCR
platform.
• To obtain optimal results, diagenode suggests some appropriate extraction methods to
avoid as much as possible PCR inhibitors (see point II. Sample Extraction/DNA Isolation).
• Extraction and/or inhibition control must be used for each reaction of PCR.
Caution
Diagenode commercializes a qualitative extraction and/or inhibition control. It is left to the
appreciation of the scientists/laboratory technicians to change the extraction procedure
according to the result obtained for this extraction and/or inhibition control in order to get an
appropriate result.
• Extraction virus control must be manipulated under safety cabinet.
Contamination
• Perform nucleic acid release, isolation and real time amplification in separate laboratory
areas.
• Keep all tubes and vials closed when not in use.
• The PCR laboratory (safety cabinet, DNA/RNA extraction platform, bench coat, etc.) must
be cleaned after each PCR experiment with appropriate solutions.
• The contaminated kits must be thrown in a specific dustbin for biological substances.
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DIAGENODE VARICELLA ZOSTER VIRUS REAL-TIME PCR USER MANUAL
• The experiments cannot be carried out by a sick scientist/laboratory technician presenting
the same symptoms as those from the required disease.
• The positive and negative PCR control must be performed with each reaction of PCR.
• Gloves must be worn.
• Aerosol resistant tips must be used for all PCR mixtures.
• Tubes must be harvested before opening.
• Do not pipette any of the materials by mouth. Do not smoke, eat or drink in areas which
specimens or kit reagents are handled.
• The experiments cannot be carried out by a pregnant woman.
Conservation
• The kit must be protected from the light.
Material Required, Not Provided
• Pipettes
• Sterile pipette tips with filters
• Powder-free gloves
• Vortex mixer
• Desktop centrifuge
• RNase/Dnase-free microcentrifuge tubes (1,5 and/or 2 ml)
• RNase/DNase free water
• DNA isolation kit (see point II. Sample Extraction/DNA Isolation)
• Consumables and accessories (reaction tubes, microcentrifuge, reaction plates,
adhesive seal) for an ABI / Roche / Biorad / Qiagen / Stratagene or Cepheid system
• Master mix (see point III. PCR Reaction Set-up)
• Real time PCR instrument: ABI / Roche / Biorad / Qiagen / Stratagene or Cepheid
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Human samples’ Collection, Storage and Transport
I. Samples’ Collection
Human samples such as bronchoalveolar lavage, tissue/biopsy, vesicular fluid, ocular fluid,
swab corneal scraping, cerebrospinal fluid, amniotic fluid, plasma and serum should be
collected.
II. Samples’ Storage
The human sample must be stored at 4°C for up to 24 hours or frozen at -20°C or -80°C for
longer periods of time.
Human samples should be stored in DNase/Rnase polypropylene tubes. The sensitivity of the
PCR could be reduced if you keep freezing and thawing human samples repeatedly.
III.Samples’ transport
Human respiratory samples must be transported in adapted containers. The samples must
be transported according to the local and national instructions for the transport of pathogen
material. Human samples should be transported at -20°C.
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DIAGENODE VARICELLA ZOSTER VIRUS REAL-TIME PCR USER MANUAL
Protocol
A. Procedures to be followed
A.1 Extraction procedure
This operation must be performed in a specific adapted room. Only this procedure could be
performed in this selected room.
Please follow the following steps:
• Place sample(s) at room temperature
NB:
It is recommended to manipulate potential infected samples under a safety cabinet.
• Chose an extracted method validated for the sample and the pathogen researched.
Diagenode selected three methods and suggests the extraction and the elution volumes
(see point II. Sample Extraction/DNA Isolation).
Please, follow manufacturer’s instructions to perform extractions.
• (Optional) add 10 μl of the diagenode DNA extraction & inhibition control (DNA virus culture
tube) in the extracted volume (see point I. DNA Extraction & inhibition control).
NB:
The diagenode DNA extraction & inhibition control is provided separately, ref: DIA-EIC/DNA-50.
You can select the dye adapted to your real-time PCR system (see point V. Selection of the
appropriate dyes).
• When the extraction procedure is finished, conserve the DNA on ice before using it (or at
- 20°C if you don’t use it the same day).
• Place also the diagenode varicella zoster virus positive control from Positive control’s
pocket on ice (see chapter Product Description).
NB:
To avoid contaminations, samples and the positive control must stay in the extraction room.
A.2 PCR procedures
This operation must be performed in a specific adapted room. Only this procedure could be
performed in this selected room.
Please follow the following steps:
PCR protocol (see point III. PCR Reaction Set-up):
Master mix suggested: Quantifast Multiplex + R kit (ref : 204756), Qiagen (Not supplied) (see
point III. PCR Reaction Set-up)
• Place the varicella zoster virus kit at room temperature: the mallow tube (varicella zoster
virus primers & probe), the dark blue tube (ddH2O) from primers & probe’s pocket (see
chapter Product Description).
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• If using DIA-EIC/DNA-050, place the tube containing primers & probe at room
temperature.
• Place the master mix on ice (qiagen, not provided).
NB:
When tubes are thawed, spin down all solutions briefly.
Before using solutions, homogenize gently by pipetting up and down.
• Select the appropriate protocol depending of the real time PCR system used (see point III.
PCR Reaction Set-up) and prepare the mix without the sample.
NB:
Prepare the same mix for all samples.
To validate the run, you need to perform a negative (water) and a positive control (diagenode
positive control) PCR.
If DIA-EIC/DNA-050 is not use, replace diagenode DNA extraction & PCR inhibition control
double-dye probe & primers by ddH2O.
• Dispense the mix prepared in appropriate tubes/capillaries or a 96 well plate.
NB:
When the mix prepared is distributed, go back to the extraction room.
• Add samples and the diagenode positive control (same volume as samples).
• Close tubes or the plate.
A.3 Amplification and real-time system procedures
This operation must be performed in a specific adapted room. Only this procedure could be
performed in this selected room.
Diagenode designs and validates kits adapted on ABI / Roche / Biorad / Qiagen / Stratagene
or Cepheid real time PCR Systems. Follow manufacturer’s instructions before using real-time
instruments.
Please follow the following steps:
• Place closed tubes or the plate in the real-time system.
• Select the PCR profile (see point IV. PCR profile).
• Select dyes adapted to the real-time system used (see point V. Selection of the appropriate
dyes):
- Varicella zoster virus (DIA-VZV-050) is detected with Fam dye.
- DNA extraction & inhibition control (DIA-EIC/DNA-050) is detected with Yellow Dye or Orange
Dye or Cy5.
• Enter the name and the position of samples in the software.
• Start the run.
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DIAGENODE VARICELLA ZOSTER VIRUS REAL-TIME PCR USER MANUAL
A.4 Interpretation of results
Follow manufacturer’s instructions for analyzing results.
Please follow the following steps:
• Analyze the FAM detector (varicella zoster virus) and the Yellow Dye/Orange Dye/Cy5
detector (DNA extraction and PCR inhibition control) separately (see point VI. Interpretation
of results):
- Negative control: FAM signal must not be observed.
- Positive control: FAM signal must be observed, Ct/Cp value around 30 (depending of the
real-time system used!).
- For Unknown sample:
• FAM signal observed indicates that the sample is positive for varicella zoster
virus.
• FAM signal not observed indicates that the sample is negative for varicella
zoster virus
If FAM signal not observed:
• Yellow Dye or Orange Dye or Cy5 signal above the threshold, 27 ≤ Ct value ≥ 36, must be
considered as positive for the DNA extraction and PCR inhibition control. The sample is
valid.
• Yellow Dye or Orange Dye or Cy5 signal below the threshold must be considered as negative
for the DNA extraction and PCR inhibition control. The sample is invalid. Scientists must
start again the DNA extraction procedure and/or the PCR procedure.
NB:
If the Ct value of the DNA extraction and PCR inhibition control (DIA-EIC/DNA-050) is ≥ 36:
I.First, we severely suggest starting again the PCR with a 10x dilution of the sample in order
to exclude as many as possible inhibition factors.
II.Secondly, if the mistake persists, we severely suggest starting again the extraction procedure
and/or to investigate another more appropriate nucleic acid extraction procedure
Select the report document to obtain a summary of Ct/Cp results and curves.
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B. Annexes of the procedure (points I-II-III-IV-V-VI):
I.DNA extraction & inhibition control (optional)
Diagenode’s DNA extraction & inhibition control is a virus culture containing a 1.000 TCID50/ml
viral load.
Universal extraction & inhibition DNA control is a complete virus, having preserved
its infectious power. This virus is listed in the Ea1* class established by the European
Federation of Biotechnologies (EFP). This virus is not reported to be dangerous for
human beings but it must be used under safety cabinet.
*(more details at the end of this manual)
The following results were obtained when using Nuclisens easyMAG® System (Biomerieux).
Procedure:
• 10 μl of each dilution extracted in 500
μl of water.
• Nuclisens easyMAG® System on
board extraction.
• Elution in 60 μl.
• 5 μl used for PCR.
• TaqMan® Universal PCR mastermix
(Applied Biosystems).
• Experiments were performed on
the ABI Prism® 7000 instruments
(Applied Biosystems).
The Ct value for 1.000 TCID50/ml is +/-28,5. The picture also shows results for other dilutions (100
TCID50/ml is +/-31,5 and 10 TCID50/ml is +/-35).
For Sample Extraction/DNA Isolation, add 10μl of the IC DNA virus culture into the sample just
before the beginning of the extraction procedure. (The concentration can be modified in order
to adapt multiplex PCRs with the extraction method used.)
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DIAGENODE VARICELLA ZOSTER VIRUS REAL-TIME PCR USER MANUAL
II. Sample Extraction/DNA Isolation
Diagenode suggests some appropriate extraction methods:
> Nuclisens easyMAG® System (Biomerieux)
Clinical samples: bronchoalveolar lavage samples, vesicular fluid, ocular fluid, cerebrospinal
fluid, plasma, serum.
Follow manufacturer’s instructions with the following:
Human sample extracted volume: 250 µl (+ 10 μl of the IC DNA virus culture)*
DNA isolation volume: 60 µl
*For Bronchoalveolar lavage: use 500 μl (+ 10 μl of the IC DNA virus culture)
The following pre-extraction procedure is recommended:
*Bronchoalveolar lavage (BAL):
• 250 μl whole sputum
• Add 250 μl proteinase K buffer (1mg/ml proteinase K, 0,5% SDS, 20 mM Tris - HCl, pH 8.3),
vortex
NB:
Proteinase K (100 mg), Merck, ref: 1.24568.0100
• Incubate 60 minutes at 55°C
• Use the complete 500 μl manipulated sputum for downstream extraction procedure
(Nuclisens Lysis Buffer)
> MagNa pure LC system, Roche: MagNa pure LC Total Nucleic Acid Isolation Kit (Ref.
03038505001)
Clinical samples: bronchoalveolar lavage, vesicular fluid, ocular fluid, swabs corneal scraping,
cerebrospinal fluid, amniotic fluid, plasma, serum, tissue/biopsy.
Follow manufacturer’s instructions with the following:
Human sample extracted volume: 200 µl (+ 10 μl of the IC DNA virus culture)
DNA elution volume: 100 µl
The following pre-extraction procedure is recommended:
Tissue/biopsy:
• Tissue/biopsy are incubated overnight at 60°C under agitation with a mixture of 0,2 mg/ml
of proteinase K, 0,5% Tween 20 and 20 mM Tris-HCl (pH 8,3).
NB:
Proteinase K (100 mg), Merck, ref: 1.24568.0100
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Bronchoalveolar lavage (BAL):
• Add 3 volume of sputolysin (1:10) with 1 volume of the BAL sample, vortex
NB:
Sputolysin, Calbiochem, ref: 560000
Before using sputolysin, the solution must be diluted 10X with DNA/RNAse water.
• Incubate 15 minutes at room temperature
• Centrifuge 5 minutes at 1500 rpm
• Completely remove and discard the supernatant
• The pellet is completely resuspended with 200 µl of sterile TE buffer
> QIAamp UltraSens Virus Kit (50) (Quiagen Ref. 53704)
Clinical samples: plasma, serum, vesicular fluid, ocular fluid, cerebrospinal fluid
Follow manufacturer’s instructions with the following:
Human sample extracted volume: 200 µl (+ 10 μl of the IC DNA virus culture)
DNA isolation volume: 60 µl
General extraction information:
If you perform the PCR on the same day, the DNA extraction can be conserved on ice. For a
longer conservation period, DNA must be stored at -20°C.
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DIAGENODE VARICELLA ZOSTER VIRUS REAL-TIME PCR USER MANUAL
III. PCR Reaction Set-up
(ABI 7000-7300-7500-7900/ Roche Lc480-Lc2.0 / Biorad iCycler-IQ5-CFX96 / Stratagene MX3000P3500P / Qiagen Rotor-Gene / Cepheid SmartCycler II)
With Quantifast Multiplex + R kit (ref : 204756), Qiagen (Not supplied)
• PCR 50 µl:
(ABI 7000-7300-7500-7900/ Roche Lc480 / Biorad iCycler-IQ5 / Stratagene MX3000P3500P / Qiagen Rotor-Gene)
25 µl.................................... 2X master mix
5 µl...................................... Diagenode varicella zoster virus double-dye probes & primers
5 µl...................................... Diagenode DNA extraction & PCR inhibition control double-dye probe &
primers
10 µl.................................... DNA
5 µl...................................... Diagenode water
50 µl.................................... Final volume
NB:
With ABI system, you can add 1 µl of the ROX solution (Qiagen).
• PCR 25 µl:
(ABI 7000-7300-7500-7900/ Roche Lc480 / Biorad iCycler-IQ5-CFX 96 / Stratagene
MX3000P-3005P / Qiagen Rotor-Gene / Cepheid SmartCycler II)
12.5 µl................................. 2X master mix
2.5 µl................................... Diagenode varicella zoster virus double-dye probes & primers
2.5 µl................................... Diagenode DNA extraction & PCR inhibition control double-dye probe &
primers
5 µl...................................... DNA
2.5 µl................................... Diagenode water
25 µl.................................... Final volume
NB:
With ABI system, you can add 1 µl of the ROX solution (Qiagen).
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• PCR 20 µl
(Roche Lc2.0)
NB:
For LightCycler 2.0 (Lc2.0), we specially suggested LightCycler® TaqMan® Master, Roche
(Not supplied) (ref: 04535286001)
4 µl......................................
5X master mix
2 µl......................................
Diagenode varicella zoster virus double-dye probes & primers
2 µl......................................
Diagenode DNA extraction & PCR inhibition control double-dye probe &
primers
10 µl....................................
Human sample (or varicella zoster virus positive control)
2 µl......................................
Diagenode water
20 µl....................................
Final volume
Caution
A new mixture must be freshly prepared for each new PCR run. Master mix (not supplied) and
primers/probes supplied in the kit must be placed on ice during the whole PCR preparation.
IV. PCR profile
Stage
Description
1
2 minutes at 50°C (1 cycle)
2
10 minutes at 95°C (1 cycle)
3
Cycle program (45 cycles)
Step 1: 15 seconds at 95°C
Step 2: 60 seconds at 60°C
NB:
For the step 2 from the stage 3, it is highly recommended to decrease the ramping rate
(°C/s) by 50%.
• Roche Lc2.0
Stage
Description
1
10 minutes at 95°C (1 cycle)
2
Cycle program (45 cycles)
Step 1: 10 seconds at 95°C
Step 2: 40 seconds at 60°C
Step 3: 1 second at 72°C
3
30 seconds at 40°C (1 cycle)
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PAGE 16
DIAGENODE VARICELLA ZOSTER VIRUS REAL-TIME PCR USER MANUAL
V. Selection of the appropriate dyes
> ABI 7000-7300-7500-7900
Detector’s name
Reporter
Quencher
Varicella zoster virus
FAM
FAM
(none)
DIA-DNA/EIC(YD)-050
Yellow Dye
Vic
(none)
DIA-DNA/EIC(DR)-050
Orange Dye
NED
(none)
DIA-DNA/EIC(Cy5)-050
Cy5
Cy5
(none)
Select Passive Reference: ROX (if using master mix with ROX reference).
Select Passive Reference: none (if using master mix without ROX reference)
DIA-DNA/EIC(YD)-050: ABI 7000 or 7300
DIA-DNA/EIC(DR)-050: ABI 7000 or 7300
DIA-DNA/EIC(Cy5)-050: ABI 7500 or 7900
> Roche Lc480-Lc2.0
Detector’s name
Reporter
Quencher
Varicella zoster virus
FAM
FAM
(none)
DIA-DNA/EIC(YD)-050
Yellow Dye
Vic/Hex
(none)
DIA-DNA/EIC(Cy5)-050
Cy5
Cy5
(none)
DIA-DNA/EIC(YD)-050: Lc2.0
DIA-DNA/EIC(Cy5)-050: Lc480
NB:
Before using our real time PCR kit for the first time on the roche lightcycler system 480 or 2.0, you
must create an application-specific color compensation object.
> Biorad iCycler-IQ5-CFX96
Detector’s name
Reporter
Quencher
Varicella zoster virus
FAM
FAM
(none)
DIA-DNA/EIC(Cy5)-050
Cy5
Cy5
(none)
DIA-DNA/EIC(Cy5)-050: iCycler or IQ5 or CFX96
Diagenode sa / LIEGE SCIENCE PARK // Rue Bois Saint-Jean, 3 // 4102 Seraing (Ougrée) // Belgium // Phone: (+32) 4 364 20 50 // Mail: [email protected]
PAGE 17
> Stratagene MX3000P-3500P
Detector’s name
Reporter
Quencher
Varicella zoster virus
FAM
FAM
(none)
DIA-DNA/EIC(Cy5)-050
Cy5
Cy5
(none)
NB:
In Filter Set Gain Settings:
• FAM: select x8
• Cy5: select x4
> Qiagen Rotor-Gene
Detector’s name
Channel
Varicella zoster virus
FAM
Green
DIA-DNA/EIC(Cy5)-050
Cy5
Red
NB:
Perform an optimization at 60 degrees at the beginning of each run for the green and red
channels.
> Cepheid SmartCycler II
Detector’s name
Reporter
Quencher
Varicella zoster virus
FAM
FAM
(none)
DIA-DNA/EIC(TR)-050
Texas red
Texas red
(none)
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PAGE 18
DIAGENODE VARICELLA ZOSTER VIRUS REAL-TIME PCR USER MANUAL
VI. Interpretation of results
Signal detection
Signal detection
FAM
Yellow dye
Interpretation of results
Orange dye
Texas red
Cy5
Signal is above the threshold
Signal is above the threshold
The sample is positive for
varicella zoster virus
Signal is above the threshold
Signal is below the threshold
The sample is positive for
varicella zoster virus
Signal is below the threshold
Signal is above the threshold
The sample is negative for
varicella zoster virus
Signal is below the threshold
Signal is below the threshold
The sample is invalid.
See Troubleshooting Guide
Diagenode sa / LIEGE SCIENCE PARK // Rue Bois Saint-Jean, 3 // 4102 Seraing (Ougrée) // Belgium // Phone: (+32) 4 364 20 50 // Mail: [email protected]
PAGE 19
Troubleshooting guide
> Nucleic Acid isolation
Refer to the manufacturer’s product insert
> PCR
The signal detected for DNA extraction and/or inhibition control is negative or weak:
• The PCR conditions do not comply with the protocol. Check the PCR conditions.
• The PCR reaction was inhibited. Check the isolation method.
• The storage conditions for the kit did not comply with recommendations.
> Real time PCR system
Refer to the manufacturer’s product insert
For trouble shootings/questions, please contact us:
Tel.: +32(0)43642050 - [email protected]
Specifications
I. Sensitivity
Determination of the sensitivity was performed on a varicella zoster virus DNA control, Vircell.
The PCR was done as described previously (see point IV PCR reaction set-up, with PCR inhibition
control). A dilution was interpreted as positive if the relative fluorescence crossed the threshold
as determined by the Biorad iCycler detection system software.
The following results were obtained:
Organism
Limit of detection
Strain
ATTC
VZV
50 copies/PCR reaction
Ellen
VR-1367
II. Specificity
The specific primers and probes were checked for possible homologies to other known sequences
by sequence comparison analysis. Primers and probes are used routinely in clinical diagnostic
laboratories on bronchoalveolar lavage, vesicular fluid, ocular fluid, swabs corneal scraping,
cerebrospinal fluid, amniotic fluid, plasma, serum, tissue/biopsy. Internal control sequence has
also been checked by sequence analysis.
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PAGE 20
DIAGENODE VARICELLA ZOSTER VIRUS REAL-TIME PCR USER MANUAL
Cross-reaction tests have been performed with the following viruses:
Herpes simplex 1
Herpes simplex 2
Epstein-Barr virus
Cytomegalovirus
Herpes virus 6
Herpes virus 7
Herpes virus 8
III.Diagnostic evaluation
An evaluation was done with Diagenode’s varicella zoster virus Real-Time PCR with around 2083
clinical samples:
• Samples were extracted with the MagNa pure LC system, Roche (MagNa pure LC Total
Nucleic Acid Isolation Kit).
• Biorad iCycler real-time PCR system.
Results were validated by an accredited laboratory.
Real-time PCRs was interpreted as positive if the relative fluorescence crossed the threshold as
determined by the Biorad iCycler detection system’s software.
The following results were obtained:
Sample
Number of samples
VZV
Plasma/serum
398
140
Cerebrospinal fluid
785
26
Swab
565
143
Tissue/biopsy
26
3
Vesicular fluid
218
85
Swab corneal scraping
20
0
Ocular fluid
24
1
Amniotic fluid
3
0
Bronchoalveolar lavage
44
6
Diagenode sa / LIEGE SCIENCE PARK // Rue Bois Saint-Jean, 3 // 4102 Seraing (Ougrée) // Belgium // Phone: (+32) 4 364 20 50 // Mail: [email protected]
PAGE 21
Product Use Limitations
• All reagents must exclusively be used for in vitro diagnostics.
• A strict compliance with the user manual is required for optimal results.
• Reliable results are dependent on adequate specimen collection, transport, storage and
processing procedures.
• This test has been validated for use with with bronchoalveolar lavage, vesicular fluid,
ocular fluid, swabs corneal scraping, cerebrospinal fluid, amniotic fluid, plasma, serum,
tissue/biopsy.
• This test has been validated for use with the ABI 7000-7300-7500-7900/ Roche Lc480Lc2.0 / Biorad iCycler-IQ5-CFX96 / Stratagene MX3000P-3500P / Qiagen Rotor-Gene /
Cepheid SmartCycler II real Time PCR systems.
Quality Control
Diagenode has obtained ISO 9001 and ISO 13485 certifications for the design, the production and
the sale of clinical diagnostic kits based on the real time PCR technology.
Varicella zoster virus Real-Time PCR is tested against predetermined specifications to ensure the
product quality.
References
(1)Steiner I, Kennedy PG, Pachner AR (2007). "The neurotropic herpes viruses: herpes simplex
and varicella-zoster". Lancet Neurol 6 (11): 1015–28.
(2)Adam Meijer, Antoine Beerens, Eric Claas at al. (2009), "Preparing the outbreak assistance
laboratory network in the Netherlands for the detection of the influenza virus A(H1N1)
variant.", J Clin Virology 45: 179-84.
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PAGE 22
DIAGENODE VARICELLA ZOSTER VIRUS REAL-TIME PCR USER MANUAL
Explanation of Symbols
Catalogue number
Batch code
Number of experiments
Temperature limitation
yyyy-mm
Expiry date
In vitro diagnostic medical device
Refer to user manual
Biological risk
Control
Negative control
Positive control
Protected from light
Manufacturer
Diagenode sa / LIEGE SCIENCE PARK // Rue Bois Saint-Jean, 3 // 4102 Seraing (Ougrée) // Belgium // Phone: (+32) 4 364 20 50 // Mail: [email protected]
PAGE 23
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.
• ABI (Applied Biosystems) Prism 7000 or 7300 or 7500 or 7900 are a trademark of Applera
Corporation
• Roche Lightcycler 480 and 2.0 are a trademark of Roche
• Biorad iCycler or IQ5 or CFX96 are a trademark of Biorad
• Stratagene MX3000P or 3500P are a trademark of Stratagene
• Qiagen Rotor-Gene is a trademark of Qiagen
• Cepheid SmartCycler II is a trademark of Cepheid
• Nuclisens easyMAG® System is a trademark of Biomerieux
• MagNa pure LC system is a trademark of Roche
• QIAamp UltraSens Virus Kit (50) is a trademark of Qiagen
• Quantifast Multiplex+R kit is a trademark of Qiagen
MA-VZV-V1_27_08_14
* Class Ea1: virus which can cause diseases to animals, and present the following
characteristics (with variable degrees): limited geographic importance, weak
interspecies transmissibility, vector or bearer non-existent. It does not normally
require particular measures of seclusion. There is usually disease prevention and/
or an effective treatment.
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Diagenode sa
LIEGE SCIENCE PARK
Rue Bois Saint-Jean, 3
4102 Seraing - BELGIUM
Tel. +32 4 364 20 50
[email protected]
www.diagenodediagnostics.com