DATA SHEET HIV-2 Real Time RT-PCR Kit

DATA SHEET
HIV-2 Real Time RT-PCR Kit
Cat. No: SR-0021-02
For use with ABI Prism®7000/7300/7500/7900; Smart
CyclerII;iCycler iQ™4/iQ™5;
Rotor-Gene6000;Mx3000P/3005P; MJ-Option2/Chromo4;
LightCycler®480real time PCR systems
For In Vitro Diagnostic Use Only
User Manual
1. Intended Use
HIV-2 real time RT-PCR kit is used for the detection
of HIV-2 in serum by using real time PCR systems.
2. Principle of Real-Time PCR
The principle of the real-time detection is based on
the fluorogenic 5’nuclease assay. During the PCR
reaction, the DNA polymerase cleaves the probe at
the 5’ end and separates the reporter dye from the
quencher dye only when the probe hybridizes to
the target DNA. This cleavage results in the
fluorescent signal generated by the cleaved
reporter dye, which is monitored real-time by the
PCR detection system. The PCR cycle at which an
increase in the fluorescence signal is detected
initially (Ct) is proportional to the amount of the
specific PCR product. Monitoring the fluorescence
intensities during Real Time allows the detection of
the accumulating product without having to reopen the reaction tube after the amplificaton.
3. Product Description
Human immunodeficiency virus (HIV) is a
retrovirus
that
can
lead
to
acquired
immunodeficiency syndrome (AIDS). Infection with
HIV occurs by the transfer of blood, semen, vaginal
fluid, pre-ejaculate, or breast milk. HIV infection in
humans is now pandemic. Two species of HIV
infect humans: HIV-1 and HIV-2. HIV-1 is more
virulent. It is easily transmitted and is the cause of
the majority of HIV infections globally. HIV-2 is less
transmittable and is largely confined to West
Africa.
HIV-2 real time RT-PCR kit contains a specific
ready-to-use system for HIV-2 detection through
Reverse Transcription Polymerase Chain Reaction
(RT-PCR) in the real-time PCR system. The master
contains Super Mix for the specific amplification of
HIV-2 RNA including subtype A~G.
The reaction is done in one step real time RT-PCR.
The first step is a reverse transcription (RT), during
which the HIV-2 RNA is transcribed into cDNA.
Then, a thermostable DNA polymerase is used to
amplify the specific gene fragments by polymerase
chain reaction (PCR). Fluorescence is emitted and
measured by the real time systems´ optical unit
during the PCR. The detection of amplified HIV-2
DNA fragment is performed in fluorimeter channel
FAM with the fluorescent quencher BHQ1. In
addition, the kit can be used for identification of
possible PCR inhibition by measuring the
HEX/VIC/JOE fluorescence of the internal control
(IC). An external positive control defined as
7
1×10 IU/ml is supplied. It allows the determination
of the gene load. For further information, please
refer to section 9.3 Quantitation.
4. Kit Contents
Analysis sensitivity: 5~103IU/ml; LOQ: 1~104`1~108IU/ml
5. Storage
• All reagents should be stored at -20°C. Storage at
+4°C is not recommended.
• All reagents can be used until the expiration date
indicated on the kit label.
• Repeated thawing and freezing (>3x) should be
avoided, as this may reduce the sensitivity of the
assay.
• Cool all reagents during the working steps.
• Super Mix should be stored in the dark.
6. Additionally Required Materials and Devices
• Biological cabinet
• Real time PCR system
• Desktop microcentrifuge for “eppendorf” type
tubes (RCF max. 16,000 x g)
• Vortex mixer
• RNA extraction kit
• Real time PCR reaction tubes/plates
• Cryo-container
• Pipets (0.5μl – 1000μl)
• Sterile filter tips for micro pipets
• Sterile microtubes
• Disposable gloves, powderless
• Biohazard waste container
• Refrigerator and Freezer
• Tube racks
Gentaur Molecular Products
Voortstraat 49
1910 Kampenhout, Belgium
7. Warnings and Precaution
Carefully read this instruction before starting the
procedure.
• For in vitro diagnostic use only.
• This assay needs to be carried out by skilled
personnel.
• Clinical samples should be regarded as
potentially infectious materials and should be
prepared in a laminar flow hood.
• This assay needs to be run according to Good
Laboratory Practice.
• Do not use the kit after its expiration date.
• Avoid repeated thawing and freezing of the
reagents, this may reduce the sensitivity of the
test.
• Once the reagents have been thawed, vortex and
centrifuge briefly the tubes before use.
• Prepare quickly the Reaction mix on ice or in the
cooling block.
• Set up two separate working areas: 1) isolation of
the RNA/ DNA and 2)
amplification/detection of amplification products.
• Pipets, vials and other working materials should
not circulate among working units.
• Use always sterile pipette tips with filters.
• Wear separate coats and gloves in each area.
• Avoid aerosols
8. Sample Collection, Storage and transport
• Collected samples in sterile tubes;
• Specimens can be extracted immediately or
frozen at -20°C to -80°C.
• Transportation of clinical specimens must comply
with local regulations for the transport of etiologic
agents.
9. Procedure
9.1 RNA-Extraction
Different brand RNA Extraction kits are available.
You may use your own extraction systems or the
commercial kit based on the yield. For the RNA
extraction, please comply with the manufacturer’s
instructions. The recommended extraction kit is as
follows:
molecular grade water before detection, and close
the tube immediately then vortex for 10 seconds.
Because of transportation with carbon dioxide ice,
there may be white precipitate in tubes of internal
control and positive control, but it will disappear in
a few minutes when it is incubated at room
temperature. Besides, the white precipitate have
no effection on the detection result.
9.3 Quantitation
The kit can be used for quantitative or qualitative
real-time RT-PCR. A positive control defined
7
as 1×10 IU/ml is supplied in the kit.
For performance of quantitative real-time PCR,
standard dilutions must be prepared first as
follows. Molecular Grade Water is used for
dilution.
The step of dilution is not needed for performance
of qualitative real-time PCR.
7
Take positive control (1×10 IU/ml) as the starting
high standard in the first tube. Respectively pipette
36ul of Molecular Grade Water into next three
tubes. Do three dilutions as the following figures:
To generate a standard curve on the real-time
system, all four dilution standards should be used
and defined as standard with specification of the
corresponding concentrations.
Attention:
A. Mix thoroughly before next transfer.
7
B. The positive control (1×10 IU/ml) contains high
concentration of the target DNA. Therefore, be
careful during the dilution in order to avoid
contamination.
9.4 RT-PCR Protocol
The Master Mix volume for each reaction should
be
pipetted
as
follows:
9.2 Internal Control and Positive Control
It is necessary to add internal control (IC) in the
reaction mix. Internal Control (IC) allows the user
to determine and control the possibility of PCR
inhibition.
Add the internal control (IC) 1μl/rxn and the result
will be shown in the HEX/VIC/JOE.
Attention: It is necessary to dilute the internal
control supplied in the kit by 10 times with
Gentaur Molecular Products
Voortstraat 49
1910 Kampenhout, Belgium
※PCR system without HEX/VIC/JOE channel may be treated
with 1μl Molecular Grade Water instead of 1μl IC.
1) The volumes of Super Mix and Enzyme Mix per
reaction multiply with the number of samples,
which includes the number of controls, standards,
and sample prepared. Molecular Grade Water is
used as the negative control. For reasons of
unprecise pipetting, always add an extra virtual
sample. Mix completely then spin down briefly in a
centrifuge.
2) Pipet 20μl Master Mix with micropipets of
sterile filter tips to each of the Real time PCR
reaction plate/tubes. Separately add 5μl RNA
sample, positive and negative controls to different
reaction plate/tubes. Immediately close the
plate/tubes to avoid contamination.
3) Spin down briefly in order to collect the Master
Mix in the bottom of the reaction tubes.
4) Perform the following protocol in the
instrument:
45°C for 10 min, 1 cycle;95°C for 15 min,
1 cycle;95°C for 15 sec, 60°C for 60sec, 40 cycles.
Fluorescence is measured at 60°C;FAM and
HEX/VIC/JOE channels should be chosen.
5) If you use ABI Prism® system, please choose
“none” as passive reference and quencher.
Control appears. The sample does not contain any
HIV-2 RNA. It can be considered negative.
4) Neither in channel FAM nor in channel
HEX/VIC/JOE is a signal detected. A diagnostic
statement can not be made. Inhibition of the RTPCR reaction.
10. Baseline setting:
just above the maximum level of molecular grade
water.
11.Calabration for quantitative detection:
Input each concentration of standard controls at
the end of run, and a standard curve will be
automatically formed.
12.Quality control:
The Ct value of molecular grade water and positive
control in FAM channel shows UNDET and ≤35
respectively; The Ct value of internal control in
HEX/VIC/JOE channel shows 25~33; Correlation
coefficient of standard curve should be ≤-0.98,
otherwise the result is invalid.
13. Data Analysis and Interpretation
The following results are possible:
1) The Ct value in channel FAM shows ≤38. The
result is positive: The sample contains HIV-2 RNA.
2) The Ct value in channel FAM shows 38~40,
please repeat again. If the result still shows
38~40,it can be considered negative.
3) In channel FAM no signal is detected, at the
same time, a HEX/VIC/JOE signal from the Internal
Gentaur Molecular Products
Voortstraat 49
1910 Kampenhout, Belgium
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