Revision No.: ZJ0003 Issue Date: Jul 1st, 2012 Ebola Virus (EBOV) Real Time RT-PCR Kit User Manual |
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For In Vitro Diagnostic Use Only QR-0220-02 |
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For use with ABI Prism®7000/7300/7500/7900/Step One Plus; iCycler iQ™4/iQ™5; Smart Cycler II;Bio-Rad CFX 96;Rotor Gene™6000; Mx3000P/3005P;MJ-Option2/Chromo4; LightCycler®480 Instrument |
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? Cool all reagents during the working steps.
? Super Mix should be stored in the dark.
6. Additionally Required Materials and Devices
? Biological cabinet ? Vortex mixer ? Cryo-container ? Sterile filter tips for micro pipets ? Disposable gloves, powderless ? Refrigerator and Freezer |
? Real time PCR system ? Real time PCR reaction tubes/plates ? Pipets (0.5μl - 1000μl) ? Sterile microtubes ? Biohazard waste container ? Tube racks |
? Desktop microcentrifuge for "eppendorf" type tubes (RCF max. 16,000 x g) |
7. Warnings and Precaution 1. Intended Use
Ebola Virus (EBOV) real time RT-PCR kit is used for the detection of EBOV in blood,serum,plasma(non-heparin anticoagulant)by using the 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 is proportional to the amount of the specific PCR product. Monitoring the fluorescence intensities in real-time allows the detection of the accumulating product without having to re-open the reaction tube after the amplification.
3. Product Description
Ebola is the virus Ebolavirus (EBOV), a viral genus, and the disease Ebola hemorrhagic fever (EHF), a viral hemorrhagic fever (VHF). The virus is named after the Ebola River Valley in the Democratic Republic of the Congo (formerly Zaire), which is near the site of the first recognized outbreak in 1976 at a mission hospital run by Flemish nuns. It remained largely obscure until 1989 when several widely publicized outbreaks occurred among monkeys in the United States.
The virus interferes with the endothelial cells lining the interior surface of blood vessels and with coagulation. As the blood vessel walls become damaged and destroyed, the platelets are unable to coagulate, patients succumb to hypovolemic shock. Ebola is transmitted through bodily fluids, while conjunctiva exposure may also lead to transmission.
EBOV real time RT-PCR kit contains a specific ready-to-use system for the detection of EBOV by RT-PCR (Reverse Transcription Polymerase Chain Reaction) in the real-time PCR system. The master contains a Super Mix for the specific amplification of the EBOV RNA. The reaction is done in one step real time RT-PCR. The first step is a reverse transcription (RT), during which the EBOV RNA is transcribed into cDNA. Afterwards, a thermostable DNA polymerase is used to amplify the specific gene fragments by means of PCR (polymerase chain reaction). Fluorescence is emitted and measured by the real time systems´ optical unit during the PCR. The detection of amplified EBOV DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1. In addition, the kit contains a system to identify possible PCR inhibition by measuring the HEX/VIC/JOE fluorescence of the internal control (IC). An external positive control defined as 1×107copies/ml is supplied which allow the determination of the gene load. For further information, please refer to section 9.3 Quantitation.
4. Kit Contents
Ref. |
Type of reagent |
Presentation 25rxns |
1 2 3 4 5 |
EBOV Super Mix RT-PCR Enzyme Mix Molecular Grade Water Internal Control (IC) EBOV Positive Control (1×107copies/ml) |
1 vial, 480ml 1 vial, 28ml 1 vial, 400μl 1 vial, 30μl 1 vial, 30μl |
Analysis sensitivity: 5×103 copies/ml LOQ: 1×104~1×108 copies/ml
Note: Analysis sensitivity depends on the sample volume, elution volume, nucleic acid extraction methods and other factors .If you use the RNA extraction kits recommended, the analysis sensitivity is the same as it declares. However, when the sample volume is dozens or even hundreds of times greater than elution volume by some concentrating method, it can be much higher.
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.
? 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.
? Do not pipette by mouth. Do not eat, drink, smoke in laboratory.
? 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:
Nucleic Acid Isolation Kit |
Cat. Number |
Manufacturer |
Nucleic Acid Magnetic Extraction Kit |
ME-0000 |
ZJ Biotech |
RNA Isolation Kit |
ME-0010 |
ZJ Biotech |
QIAamp DSP Virus Spin Kit |
61704 |
QIAGEN |
QIAamp Viral RNA Mini Kit |
52904 |
QIAGEN |
9.2 Internal 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.
9.3 Quantitation
The kit can be used for quantitative or qualitative real-time RT-PCR. A positive control defined as 1×107copies/ml is supplied in the kit.
For performance of quantitative real-time PCR, Standard dilutions must prepare first as follows. Molecular Grade Water is used for dilution.
Dilution is not needed for qualitative real-time PCR detection.
Take positive control (1×107copies/ml) as the starting high standard in the first tube. Respectively pipette 36ul 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.
B. The positive control (1×107copies/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:
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 supernatantor 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:
5) Perform the following protocol in the instrument:
45°C for 10min |
1cycle |
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Selection of fluorescence channels |
95°C for 15min |
1cycle |
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FAM |
Target Nucleic Acid |
95°C for 15sec, 60°C for 1min ( Fluorescence measured at 60°C) |
40cycles |
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HEX/VIC/JOE |
IC |
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5) If you use ABI Prism® system, please choose "none" as passive reference and quencher.
10. Threshold setting: just above the maximum level of molecular grade water.
11. Calibration 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: Negative control, positive control, internal control and QS curve must be performed correctly, otherwise the sample results is invalid.
Channel Control |
Ct value |
FAM |
HEX/VIC/JOE |
Molecular Grade Water |
UNDET |
25~35 |
Positive Control(qualitative assay) |
≤35 |
-- |
QS(quantitative detection) |
Correlation coefficient of QS curve≤-0.98 |
13. Data Analysis and Interpretation
The following sample results are possible:
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Ct value |
Result Analysis |
FAM |
HEX/VIC/JOE |
1# |
UNDET |
25~35 |
Below the detection limit or negative |
2# |
≤38 |
-- |
Positive; and the software displays the quantitative value |
3# |
38~40 |
25~35 |
Re-test; if it is still 38~40, report as 1# |
4# |
UNDET |
UNDET |
PCR Inhibition; no diagnosis can be concluded. |