Infectious Hypodermal and Hematopoietic Necrosis Virus Real Time
Contents
1. 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 Control appears The sample does not contain any infectious hypodermal and hematopoietic necrosis virus DNA 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 PCR reaction 1910 Kampenhout Belgium2. gt CENTATTR DATA SHEET Infectious Hypodermal and Hematopoietic Necrosis Virus Real Time PCR Kit Cat No AD 0120 01 For Use with LightCycler 1 0 LightCycler2 0 Real Time PCR Systems For In Vitro Diagnostic Use Only User Manual 1 Intended Use Infectious Hypodermal and Hematopoietic Necrosis Virus real time PCR kit is used for the detection of Infectious Hypodermal and Hematopoietic Necrosis Virus in gill or muscle samples of Shrimp 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 re open the reaction tube after the amplification 3 Product Description Infectious hypodermal hematopoietic necrosis virus IHHNV is a small 22nm average diameter single stranded DNA containing parvovirus The host range of this species appears to include a wild shrimp sp
3. ecies This species appears to be of Indo Pacific origin but is now widely distributed primarily through introductions into aquaculture facilities worldwide This species is responsible for catastrophic epidemics in aquaculture facilities worldwide and is largely responsible for temporary cessation of Mexican commercial shrimp fishing for several years This species is extremely detrimental to the shrimp farming industry and has resulted in multi million dollar losses Further spread of this disease causing virus through transplation of infected stocks will likely continue to severely impact the industry Infectious Hypodermal and Hematopoietic Necrosis Virus real time PCR kit contains a specific ready to use system for the detection of the Infectious Hypodermal and Hematopoietic Necrosis Virus by polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the Infectious Hypodermal and Hematopoietic Necrosis Virus DNA Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified Infectious Hypodermal and Hematopoietic Necrosis Virus DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit and gill or muscle samples are used for the extraction of the DNA In addition the kit contains a system to identify possible PCR inhibition by measuring the HEX VIC JOE fluorescence o
4. erformance of qualitative real time PCR detection Take positive control 1x10 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 Dilution of Standards To generate a standard curve on Aul Au Au the real time system all four e EU i S dilution standards should be used and defined as standard with specification of the corresponding concentrations Attention y A Mix thoroughly before next Y v transfer 7 6 5 aani 1X10 1X10 1X10 1 X10 copies mi B The positive control contains high concentration of the target DNA Therefore be careful during the dilution in order to avoid contamination 9 4 PCR Protocol The Master Mix volume for each reaction should be pipetted as follow 17 ul 0 4ul 1ul Reaction Mix Enzyme Mix Internal Control l en 18 4 ul Master Mix 2 ul 18 ul Extraction DNA Master Mix a Reaction Plate Tube l PCR Instrument XPCR system without HEX VIC JOE channel may be treated with 1 Molecular Grade Water instead of Il IC 1 The volumes of Reaction 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 Pip
5. et 18u Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tubes Separately add 2n1 DNA 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 37 C for 2 min 1 cycle 94 C for 2 min 1 cycle 93 C for 5 sec 60 C for 30 sec 40 cycles Fluorescence is measured at 60 C channel FAM and HEX VIC JOE should be chosen 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 crossing point value of molecular grade water and positive control in FAM channel shows blank and lt 35 respectively The crossing point value of internal control in HEX VIC JOE channel shows 25 33 Correlation coefficient of standard curve should be lt 0 98 otherwise the result is invalid 13 Data Analysis and Interpretation The following results are possible 1 The crossing point value in channel FAM shows lt 38 The result is positive The sample contains infectious hypodermal and hematopoietic necrosis virus DNA 2 The crossing point value in channel FAM shows 38 40 please repeat again If the result still
6. f the internal control IC An external positive control 1x10 copies ml contained allows the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents DNA Extraction Buffer 1 vial 1 8ml IHHNV Reaction Mix 1 vial 450ul 1 vial 12ul 1 vial 400u1 1 vial 30ul 1 vial 30ul PCR Enzyme Mix Molecular Grade Water Internal Control IC IHNNV Positive Control 1x10 copies ml 5 Storage e All reagents should be stored at 20 C Storage at 4 C is not recommended e All reagents can be used until the expiration date indicated on the kit label e Repeated thawing and freezing gt 3x should be avoided as this may reduce the sensitivity of the assay e Cool all reagents during the working steps e Super Mix should be stored in the dark 6 Additionally Required Materials and Devices e Biological cabinet e Real time PCR system e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g e Vortex mixer e RNA extraction kit e Real time PCR reaction tubes plates e Cryo container e Pipets 0 5 ul 1000 ul e Sterile filter tips for micro pipets e Sterile microtubes e Disposable gloves powderless e Biohazard waste container e Refrigerator and Freezer e Tube racks 7 Warnings and Precaution e Carefully read this instruction before starting the procedure e For in vitro diagnostic use only e This assay needs to be carried out by skilled personnel e Clinica
7. in down briefly in a table centrifuge 2 Incubate the tube for 10 minutes at 100 C 3 Centrifuge the tube at 13000rpm for 10 minutes The supernatant contains the DNA extracted and can be used for PCR template Attention A During the incubation make sure the tube is not open as the vapor will volatilize into the air and may cause contamination in case the sample is positive B The extraction sample should be used in 3 hours or stored at 20 C for one month C DNA extraction kits are available from various manufacturers You may use your own extraction systems or the commercial kit based on the yield For DNA extraction please comply with the manufacturer s instructions 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 1ul rxn and the result will be shown in the HEX VIC JOE Attention It is necessary to dilute the positive control supplied in the kit to 10 copies ml by 10 times with molecular grade water before detection and close the tube immediately then vortex for 10 seconds 9 3 Quantitation The kit can be used for quantitative or qualitative real time PCR For performance of quantitative real time PCR standard dilutions must be prepared firstly as follows Molecular Grade Water is used as the dilution Dilution is not needed for p
8. l samples should be regarded as potentially infectious materials and should be prepared in a laminar flow hood e This assay needs to be run according to Good Laboratory Practice e Do not use the kit after its expiration date e Avoid repeated thawing and freezing of the reagents this may reduce the sensitivity of the test e Once the reagents have been thawed vortex and centrifuge briefly the tubes before use e Prepare quickly the Reaction mix on ice or in the cooling block e Set up two separate working areas 1 Isolation of the RNA DNA and 2 Amplification detection of amplification products e Pipets vials and other working materials should not circulate among working units e Use always sterile pipette tips with filters e Wear separate coats and gloves in each area e Do not pipette by mouth Do not eat drink and smoke in laboratory e Avoid aerosols 8 Sample Collection Storage and Transport e Collected samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C Gentaur Molecular P Voortstraat 49 roducts e Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents 9 Procedure 9 1 DNA Extraction DNA extraction buffer is supplied in the kit please thaw the buffer thoroughly and spin down briefly in the centrifuge before use 1 Take 50mg sample to a tube add 50u1 DNA extraction buffer close the tube then vortex for 10 seconds Sp
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