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Avian Influenza virus H5N1 Real Time RT_PCR Kit *CE Marked I, II *to see for appropriate equipment see catalogue No. MXX_000069
/Product Detail : RR-0048-01 Avian Influenza virus H5N1 Real Time RT_PCR Kit *CE Marked I, II *to see for appropriate equipment see catalogue No. MXX_000069
Product name : Avian Influenza virus H5N1 Real Time RT_PCR Kit *CE Marked I, II *to see for appropriate equipment see catalogue No. MXX_000069
Catalog number : RR-0048-01
Supplier name : Liferi
Data sheet : Ask more or other datasheet now !
For In Vitro Diagnostic Use Only
For use with LightCycler1.0/2.0 Instrument
1. Intended Use
Avian influenza virus H5N1 real time RT-PCR kit is used for the detection of avian H5N1 virus in human nasal and pharyngeal secretions and bird fece by 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 re-open the reaction tube after the amplification.
3. Product Description
Highly pathogenic avian influenza (HPAI) caused by certain subtypes of influenza A virus in animal populations, particularly chickens, poses a continuing global human public health risk.
Direct human infection by an avian influenza A(H5N1) virus was first recognized during the 1997 outbreak in Hong Kong. Subsequently, human infections with avian strains of the H9 and H7 subtypes have been further documented. Avian influenza A H5 and H7 viruses can be distinguished as “low pathogenic” and “high pathogenic” forms on the basis of genetic features of the virus and the severity of the illness they cause in poultry; influenza H9 virus has been identified only in a “low pathogenicity” form. Each of these three avian influenza A viruses (H5, H7, and H9) theoretically can be partnered with any one of nine neuraminidase surface proteins; thus, there are potentially nine different forms of each subtype (e.g., H5N1, H5N2, H5N3, H5N9). Avian influenza virus H5N1 real time RT-PCR kit contains a specific ready-to-use system for the detection of the Avian H5N1 virus by Reverse Transcription Polymerase Chain Reaction(RT-PCR) in the real-time PCR system. The master contains a Super Mix for the specific amplification of Avian influenza virus RNA. The reaction is done in one step real time RT-PCR. The first step is a reverse transcription (RT), during which the Avian influenza virus RNA is transcribed into cDNA. Afterwards, a thermostable DNA polymerase is used to amplify the specific gene fragments by means of polymerase chain reaction(PCR). Fluorescence is emitted and measured by the real time systems´ optical unit during PCR. The detection of amplified Avian influenza virus DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1. In addition, the kit contains a system to identify possible PCR inhibition by measuring the 560nm fluorescence of the internal control (IC). An external positive control(1×107copies/ml) contained, allows the determination of the gene load. For further information, please refer to section 9.3 Quantitation.
4. Kit Contents
Analysis sensitivity: 5×103 copies/ml; LOQ: 1×104～1×108copies/ml.
• 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
• Pipets (0.5 μl – 1000 μl)
• Sterile filter tips for micro pipets
• Sterile microtubes
• Disposable gloves, powderless
• Biohazard waste container
• Refrigerator and freezer
• Tube racks
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.
• 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
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:
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 560nm Channel.
The kit can be used for quantitative or qualitative real-time RT-PCR.
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 (1×10 copies/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.
A. Mix thoroughly before next transfer.
B. The positive control 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 560nm 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 15μl 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:
10. Threshold setting: Choose Arithmetic as back ground and none as Noise Band method, then adjust the Noise band just above the maximum level of molecular grade water, and adjust the threshold just under the minimum of the positive control.
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: Negative control, positive control, internal control and QS curve must be performed correctly, otherwise the sample results is invalid.
13. Data Analysis and Interpretation
The following sample results are possible:
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