Influenza A B Real TM Eng ver 21032013

IVD
For in Vitro Diagnostic Use
Influenza A B Real-TM
Handbook
Real Time Amplification test for the
detection of Influenza A and B Viruses
REF V36-50FRT
REF TV36-50FRT
50
Sacace™ Influenza A B Real-TM
VER 21.03.2013
Sacace™ Influenza A B Real-TM
VER 21.03.2013
NAME
Influenza Virus A B Real-TM
INTRODUCTION
Influenza virus infection, one of the most common infectious disease, is a highly contagious
airborne disease that causes an acute febrile illness and results in variable degrees of systemic
symptoms, ranging from mild fatigue to respiratory failure and death. These symptoms
contribute to significant loss of workdays, human suffering, mortality, and significant morbidity.
Influenza results from infection with 1 of 3 basic types of influenza virus – A, B, or C – which are
classified within the family Orthomyxoviridae. These single stranded RNA viruses are
structurally and biologically similar but vary antigenically. The most common prevailing influenza
A subtypes that infect humans are H1N1 and H3N2.
INTENDED USE
Influenza Virus A B Real-TM is Real-Time amplification test for the qualitative detection of
Influenza A and B RNA in clinical specimens.
PRINCIPLE OF ASSAY
Influenza Virus A B Real-TM Test is based on three major processes: isolation of virus RNA
from specimens, reverse transcription of the RNA, Real Time amplification of the cDNA.
Influenza virus A&B detection by the polymerase chain reaction (PCR) is based on the
amplification of pathogen genome specific region using specific primers and detection via
fluorescent dyes. These dyes are linked with probes of oligonucleotides which bind specifically
to the amplified product. The real-time PCR monitoring of fluorescence intensities allows the
accumulating product detection without reopening of reaction tubes after the PCR run.
Influenza Virus A B Real-TM PCR kit is a qualitative test which contain the Internal Control
(IC). It must be used in the isolation procedure in order to control the process of each individual
sample extraction and serves also to identify possible reaction inhibition.
Sacace™ Influenza A B Real-TM
VER 21.03.2013
MATERIALS PROVIDED
Module No.1: Real Time PCR kit (V36-50FRT)
Part N° 2 – “Reverta-L ”: Reverse transcription of the RNA
•
RT-G-mix-1, 5 x 0,01 ml;
•
RT-mix, 5 x 0,125 ml;
•
Reverse transcriptase (M-MLV), 0,03 ml;
•
TE-buffer, 1,2 ml.
Contains reagents for 60 tests.
Part N° 3 – “Influenza A&B ”: Real Time amplification kit
• PCR-mix-1, 5 x 0,12 ml;
• PCR-mix-2-FRT, 0,3 ml;
• TaqF Polymerase, 0,03 ml;
• Pos cDNA Infl. A C+, 0,1 ml;
• Pos cDNA Infl. B C+, 0,1 ml
• Negative Control, 1,2 ml*;
• Internal Control RNA, 5 x 0,12 ml**;
• Internal Control DNA, 0,1 ml;
• DNA buffer, 0,5 ml;
Contains reagents for 55 tests.
*
must be used in the isolation procedure as Negative Control of Extraction.
** add 10 µl of Internal Control RNA during the RNA purification procedure directly to the sample/lysis mixture
Sacace™ Influenza A B Real-TM
VER 21.03.2013
Module No.2: Complete Real Time PCR test with RNA purification kit (TV36-50FRT)
Part N° 1 – “Ribo-Sorb”: Sample preparation
• Lysis Solution, 22,5 ml;
• Washing Solution, 20 ml;
• Sorbent, 1,25 ml.
• RNA-eluent, 5 x 0,5ml;
Contains reagents for 50 tests.
Part N° 2 – “Reverta-L ”: Reverse transcription of the RNA
•
RT-G-mix-1, 5 x 0,01 ml;
•
RT-mix, 5 x 0,125 ml;
•
Reverse transcriptase (M-MLV), 0,03 ml;
•
TE-buffer, 1,2 ml.
Contains reagents for 60 tests.
Part N° 3 – “Influenza A&B ”: Real Time amplification kit
• PCR-mix-1, 5 x 0,12 ml;
• PCR-mix-2-FRT, 0,3 ml;
• TaqF Polymerase, 0,03 ml;
• Pos cDNA Infl. A C+, 0,1 ml;
• Pos cDNA Infl. B C+, 0,1 ml
• Negative Control, 1,2 ml*;
• Internal Control RNA, 5 x 0,12 ml**;
• Internal Control DNA, 0,1 ml;
• DNA buffer, 0,5 ml;;
Contains reagents for 55 tests.
*
must be used in the isolation procedure as Negative Control of Extraction.
** add 10 µl of Internal Control RNA during the RNA purification procedure directly to the sample/lysis mixture
Sacace™ Influenza A B Real-TM
VER 21.03.2013
MATERIALS REQUIRED BUT NOT PROVIDED
Zone 1: sample preparation (only for Module No. 2):
• RNA extraction kit (Module No. 1)
• Biosafety cabinet
• Desktop microcentrifuge for “eppendorf” type tubes (RCF max. 16,000 x g); Eppendorf 5415D
or equivalent
• 60°C ± 2°C dry heat block
• Vortex mixer
• Pipettors (capacity 5-40 µl; 40-200 µl; 200-1000 µl) with aerosol barrier
• 1,5 ml polypropylene sterile tubes (Sarstedt, QSP, Eppendorf)
• Disposable gloves, powderless
• Tube racks
• 70% Ethanol (freshly prepared mixture of reagent grade 96% ethanol and distilled water)
• Acetone
• Refrigerator
• Freezer
Zone 2: RT and amplification:
• Real Time Thermalcycler
• Workstation
• Pipettors (capacity 0,5-10 µl; 5-40 µl) with aerosol barrier
• Tube racks
STORAGE INSTRUCTIONS
Influenza Virus A B Real-TM must be stored at -20°C. Store Ribo-Sorb kit at 2-25°C.The kits
can be shipped at 2-8°C for 3-4 days but should be stored at 2-8°C and -20°C immediately on
receipt.
STABILITY
Influenza Virus A B Real-TM is stable up to the expiration date indicated on the kit label. The
product will maintain performance through the control date printed on the label. Exposure to
light, heat or humidity may affect the shelf life of some of the kit components and should be
avoided. Repeated thawing and freezing of these reagents should be avoided, as this may
reduce the sensitivity.
QUALITY CONTROL
In accordance with Sacace’s ISO 13485-Certified Quality Management System, each lot is
tested against predetermined specifications to ensure consistent product quality.
Sacace™ Influenza A B Real-TM
VER 21.03.2013
WARNINGS AND PRECAUTIONS
In Vitro Diagnostic Medical Device
For In Vitro Diagnostic Use Only
The user should always pay attention to the following:
•
Lysis Solution contains guanidine thiocyanate*. Guanidine thiocyanate is harmful if
inhaled, or comes into contact with skin or if swallowed. Contact with acid releases toxic gas.
(Xn; R: 20/21/22-36/37/38; S: 36/37/39).
• Clinical specimens from suspect influenza A (H1N1) cases should be performed in a BSL2
laboratory with BSL3 practices (enhanced BSL2 conditions). Use sterile pipette tips with
aerosol barriers and use new tip for every procedure.
• Store extracted positive material (samples, controls and amplicons) away from all other reagents
and add it to the reaction mix in a separate area.
• Thaw all components thoroughly at room temperature before starting an assay.
• When thawed, mix the components and centrifuge briefly.
• Use disposable gloves, laboratory coats and eye protection when handling specimens and
reagents. Thoroughly wash hands afterwards.
• Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in laboratory work areas.
• Do not use a kit after its expiration date.
• Dispose of all specimens and unused reagents in accordance with local authorities’
regulations.
• Specimens should be considered potentially infectious and handled in a biological cabinet in
accordance with appropriate biosafety practices.
• Clean and disinfect all sample or reagent spills using a disinfectant such as 0.5% sodium
hypochlorite, or other suitable disinfectant.
• Avoid sample or reagent contact with the skin, eyes, and mucous membranes. If skin, eyes,
or mucous membranes come into contact, rinse immediately with water and seek medical
advice immediately.
• Material Safety Data Sheets (MSDS) are available on request.
• Use of this product should be limited to personnel trained in the techniques of DNA
amplification.
• The laboratory process must be one-directional, it should begin in the Extraction Area and
then move to the Amplification and Detection Areas. Do not return samples, equipment and
reagents to the area in which the previous step was performed.
Some components of this kit contain sodium azide as a preservative. Do not use
metal tubing for reagent transfer.
* Only for Module No.2
Sacace™ Influenza A B Real-TM
VER 21.03.2013
PRODUCT USE LIMITATIONS
All reagents may exclusively be used in in vitro diagnostics. Use of this product should be
limited to personnel trained in the techniques of DNA amplification (EN375). Strict compliance
with the user manual is required for optimal PCR results. Attention should be paid to expiration
dates printed on the box and labels of all components. Do not use a kit after its expiration date.
SAMPLE COLLECTION, STORAGE AND TRANSPORT
Influenza Virus A B Real-TM can analyze RNA extracted with Ribo-Sorb (REF K-2-1) from:
• nasopharyngeal swabs: swab area and place in “Eppendorf” tube with 0,5 ml of saline water
or PBS sterile (Sacace Transport medium is recommended). Agitate vigorously. Repeat the
swab and agitate in the same tube. Centrifuge at 1000g/min for 5 min. Discard the
supernatant and leave about 100 µl of solution for RNA extraction.
• aspirate, bronchial lavage, nasal wash: centrifuge at 2000 g/min for 10-15 min. If the pellet is
not visible add 10 ml of liquid and repeat centrifugation. Remove and discard the
supernatant. Resuspend the pellet in 100 µl of Saline water.
• tissue: 1,0 gr (parenchimatous organs, trachea, lung, brain) homogenized with mechanical
homogenizer or scalpel, glass sticks, teflon pestles and dissolved in 1,0 ml of saline water or
PBS sterile. Vortex vigorously and incubate 30 min at room temperature. Transfer the
supernatant into a new 1,5 ml tube;
Specimens can be stored at +2-8°C for no longer than 12 hours, or frozen at -20°C to -80°C.
Transportation of clinical specimens must comply with country, federal, state and local
regulations for the transport of etiologic agents.
RNA ISOLATION
The following isolation kits are recommended:
⇒ Ribo-Sorb- (Sacace, REF K-2-1)
⇒ Ribo-Virus spin column extraction kit (Sacace, REF K-2-C)
Please carry out the RNA extraction according to the manufacturer’s instructions. Add 10 µl of
Internal Control during the DNA isolation procedure directly to the sample/lysis mixture.
Sacace™ Influenza A B Real-TM
VER 21.03.2013
SPECIMEN AND REAGENT PREPARATION (reagents supplied with the module no.2)
1.
Lysis Solution and Washing Solution (in case of their storage at +2-8оС) should be
warmed up to 60–65оС until disappearance of ice crystals. Prepare required quantity of 1.5
ml polypropylene tubes including one tube for Negative Control of Extraction.
2.
Add to each tube 450 µl Lysis Solution and 10 µl Internal Control (IC RNA). Mix by
pipetting and incubate 5 min at room temperature.
3.
Add 100 µl of samples to the appropriate tube containing Lysis Solution and IC.
4.
Prepare Controls as follows:
•
5.
add 100 µl of C– Negative Control to the tube labeled Cneg
Vortex the tubes and centrifuge for 5 sec at 5000g. If the sample is not completely
dissolved it is recommended to re-centrifuge the tube for 1 min at a maximum speed
(12000-16000 g.) and transfer the supernatant into a new tube for RNA extraction
6.
Vortex vigorously Sorbent and add 25 µl to each tube.
7.
Vortex for 5-7 sec and incubate all tubes for 10 min at room temperature. Vortex
periodically.
8.
Centrifuge all tubes for 1 min at 10000g and using a micropipette with a plugged aerosol
barrier tip, carefully remove and discard supernatant from each tube without disturbing the
pellet. Change tips between tubes.
9.
Add 400 µl of Washing Solution to each tube. Vortex vigorously, centrifuge for 1 min at
10000g. and using a micropipette with a plugged aerosol barrier tip, carefully remove and
discard supernatant from each tube without disturbing the pellet. Change tips between
tubes.
10. Add 500 µl of Ethanol 70% to each tube.
Vortex vigorously, centrifuge for 1 min at
10000g. and using a micropipette with a plugged aerosol barrier tip, carefully remove and
discard supernatant from each tube without disturbing the pellet. Change tips between
tubes.
11. Repeat step 10.
12. Add 400 µl of Acetone to each tube. Vortex vigorously , centrifuge for 1 min at 10000g and
using a micropipette with a plugged aerosol barrier tip, carefully remove and discard
supernatant from each tube without disturbing the pellet. Change tips between tubes.
13. Incubate all tubes with open cap for 10 min at 60°C.
14. Resuspend the pellet in 40 µl of RNA-eluent. Incubate for 10 min at 60°C and vortex
periodically. Centrifuge the tubes for 2 min at maximum speed (12000-16000 g).
15. The supernatant contains RNA ready for use. The RT-PCR can be performed the same
day. If this is not possible, the RNA preparations can be stored at -80°C for up to one
month.
Sacace™ Influenza A B Real-TM
VER 21.03.2013
RT AND AMPLIFICATION
Reverse Transcription:
1. Prepare Reaction Mix: for 12 reactions, add 5,0 µl RT-G-mix-1 into the tube containing RTmix and vortex for at least 5-10 seconds, centrifuge briefly. This mix is stable for 1 month at
-20°C. Add 6 µl M-MLV into the tube with Reagent Mix, mix by pipetting, vortex for 3 sec,
centrifuge for 5-7 sec (must be used immediately after the preparation).
(If it is necessary to test less than 12 samples add for each sample (N) in the new sterile tube
10*N µl of RT-G-mix-1 with RT-mix and 0,5*N µl of M-MLV).
2. Add 10 µl of Reaction Mix into each sample tube.
3. Pipette 10 µl RNA samples to the appropriate tube. (If the Ribo-Sorb isolation kit is used as
a RNA extraction kit, re-centrifuge all the tubes with extracted RNA for 2 min at maximum
speed (12000-16000 g) and take carefully supernatant. N.B. don’t disturb the pellet, sorbent
inhibit reaction).Carefully mix by pipetting.
4. Place tubes into thermalcycler and incubate at 37oС for 30 minutes.
5. Dilute 1: 2 each obtained cDNA sample with TE-buffer (add 20 µl TE-buffer to each tube).
cDNA specimens could be stored at -20oС for a week or at -70oС during a year.
Real Time amplification:
Reaction Mix 25 µl
1. Prepare required quantity of tubes or PCR plate.
2. Prepare for each sample in the new sterile tube 10*N µl of PCR-mix-1, 5*N µl of PCR-mix2-FRT and 0,5*N of TaqF Polymerase.
3. Add 15 µl of Reaction Mix into each tube.
4. Add 10 µl of cDNA sample to appropriate tube with Reaction Mix.
5. Prepare for each panel 4 controls:
• add 10 µl of DNA-buffer to the tube labeled Amplification Negative Control;
• add 10 µl of cDNA Influenza A C+ to the tube labeled Cpos A;
• add 10 µl of cDNA Influenza B C+ to the tube labeled Cpos B;
• add 10 µl of IC DNA to the tube labeled IC DNApos;
Sacace™ Influenza A B Real-TM
VER 21.03.2013
Amplification
1. Create a temperature profile on your instrument as follows:
1
Rotor and plate type instruments
Modular type instruments
Step
1
Temperature, °С
95
95
Time
15 min
10 s
Cycles
1
Temperature, °С
95
95
2
54
25 s
10
54
72
95
3
54
72
25 s
10 s
30 s
Fluorescence
detection
25 s
72
Time
900 s
15 s
25 s
Fluorescence
detection
25 s
2
Cycles
1
42
35
1
For example SaCycler-96™ (Sacace), CFX/iQ5™ (BioRad); Mx3005P™ (Agilent), ABI® 7300/7500/StepOne Real
Time PCR (Applied Biosystems), Rotor-Gene™ 3000/6000/Q (Corbett Research, Qiagen), LineGeneK® (Bioer)
2
For example, SmartCycler® (Cepheid)
Influenza Virus A is detected on the Rox (Orange) channel, Influenza Virus B is
detected on the JOE (Yellow) channel, IC on the FAM (Green) channel.
INSTRUMENT SETTINGS
Rotor-type instruments
Channel
FAM/Green
JOE/Yellow
Rox/Orange
Calibrate/Gain
Optimisation…
from 5 Fl to 10 Fl
from 4 Fl to 8 Fl
from 4 Fl to 8 Fl
0.1
More Settings/
Outlier Removal
5%
0.1
10 %
off
0.1
10 %
off
Threshold
Slope Correct
off
Plate- or modular type instruments
The threshold line should cross only sigmoid curves of signal accumulation of positive samples
and should not cross the baseline; otherwise, the threshold level should be raised. Set the
threshold at a level where fluorescence curves are linear and do not cross curves of the
negative samples.
Sacace™ Influenza A B Real-TM
VER 21.03.2013
RESULTS ANALYSIS
1. The sample is considered to be positive for Influenza Virus A if in the channel Rox (Orange)
the value of Ct is different from zero (must be less than 33 (37 for SmartCycler). If Ct value
is more than 33 the assay should be repeated and the sample is considered to be positive in
case of result’s repeat or in case of result is less than 33.
2. The sample is considered to be positive for Influenza Virus B if in the channel Joe (Yellow)
the value of Ct is different from zero (must be less than 33 (37 for SmartCycler). If Ct value
is more than 33 (37 for SmartCycler) the assay should be repeated and the sample is
considered to be positive in case of result’s repeat or in case of result is less than 33.
3. The sample is considered to be negative for Influenza A/B if in the channel Fam (Green)
value is not determined (the fluorescence curve does not cross the threshold line) and in the
results table on the channel Fam (Green) the Ct value is lower than 28.
Table. Results for controls
Stage for
Control
control
RNA
NCE
isolation
NCA
PCR
Pos cDNA Infl. A
PCR
Pos cDNA Infl. B
PCR
IC DNA
PCR
Ct channel
Fam (Green)
Ct channel
Joe (Yellow)
Ct channel
Rox (Orange)
Result
Pos (< 28)
Neg
Neg
Valid result
Neg
Neg
Neg
Pos (< 28)
Neg
Neg
Pos (< 33)
Neg
Neg
Pos (< 33)
Neg
Neg
Valid result
Valid result
Valid result
Valid result
PERFORMANCE CHARACTERISTICS
The kit Influenza A B Real-TM allows to detect Influenza A&B viruses in 100% of the tests with
a sensitivity of not less than 500 copies/ml.
Sacace™ Influenza A B Real-TM
VER 21.03.2013
TROUBLESHOOTING
1. Weak or absent signal of the IC (Fam (Green) channel): retesting of the sample is required.
•
The PCR was inhibited.
⇒ Make sure that you use a recommended RNA extraction method and follow
the manufacturer’s instructions.
⇒ Re-centrifuge all the tubes before pipetting the extracted RNA for 2 min at
maximum speed (12000-16000 g) and take carefully supernatant. Don’t
disturb the pellet, sorbent inhibit reaction.
•
The reagents storage conditions didn’t comply with the instructions.
⇒ Check the storage conditions
•
The PCR conditions didn’t comply with the instructions.
⇒ Check the PCR conditions and for the IC detection select the fluorescence
channel reported in the protocol.
•
The IC was not added to the sample during the pipetting of reagents.
⇒ Make attention during the RNA extraction procedure.
2. Weak signal on the Joe (Yellow)/Cy3/HEX and Rox/TexasRed channels: retesting of the
sample is required.
3. Joe (Yellow)/Cy3/HEX or Rox/TexasRed signal with Negative Control of extraction.
•
Contamination during RNA extraction procedure. All samples results are invalid.
⇒ Decontaminate all surfaces and instruments with sodium hypochlorite and
ethanol.
⇒ Use only filter tips during the extraction procedure. Change tips among
tubes.
⇒ Repeat the RNA extraction with the new set of reagents.
4. Any signal with Negative PCR Control.
•
Contamination during PCR preparation procedure. All samples results are invalid.
⇒ Decontaminate all surfaces and instruments with sodium hypochlorite and
ethanol or special DNA decontamination reagents.
⇒ Pipette the Positive controls at the end.
⇒ Repeat the PCR preparation with the new set of reagents.
Sacace™ Influenza A B Real-TM
VER 21.03.2013
Sacace™ Influenza A B Real-TM
VER 21.03.2013
Sacace™ Influenza A B Real-TM
VER 21.03.2013
KEY TO SYMBOLS USED
List Number
Caution!
Lot Number
Contains sufficient
for <n> tests
For in Vitro Diagnostic
Use
Version
Store at
NCA
Negative Control of
Amplification
Manufacturer
NCE
Negative control of
Extraction
Consult instructions for
use
C+
Positive Control of
Amplification
Expiration Date
IC
Internal Control
* SaCycler™ is a registered trademark of Sacace Biotechnologies
* CFX™ and iQ5™ are registered trademarks of Bio-Rad Laboratories
* Rotor-Gene™ is a registered trademark of Qiagen
* MX3005P® is a registered trademark of Agilent Technologies
* ABI® is a registered trademark of Applied Biosystems
* LineGeneK® is a registered trademark of Bioer
* SmartCycler® is a registered trademark of Cepheid
Sacace Biotechnologies Srl
via Scalabrini, 44 – 22100 – Como – Italy Tel +390314892927 Fax +390314892926
mail: [email protected] web: www.sacace.com
Sacace™ Influenza A B Real-TM
VER 21.03.2013