(NiV) Real Time RT-PCR Kit
Contents
1. 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 during real time allows the detection of the accumulating product without having to re open the reaction tube after the amplification 3 Product Description Nipah virus NiV is an emerging zoonotic virus In infected people Nipah virus causes severe illness characterized by inflammation of the brain encephalitis or respiratory diseases It can also cause severe disease in animals such as pigs resulting in significant economic losses for farmers Nipah virus is closely related to Hendra virus Both are members of the genus Henipavirus a new class of virus in the Paramyxoviridae family Although Nipah virus has caused only a few outbreaks it infects a wide range of animals and causes severe disease and death in people making it a public health concern The Nipah Virus NiV real time RT PCR Kit contains a specific ready to use sys2. 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 560nm 9 3 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 134l 1 ul ipl Super Mix Enzyme Mix Internal Control Spl 154l Extraction RNA Master Mix Reaction Plate Tube PCR Instrument XX PCR system without 560nm channel may be treated with 1u Molecular Grade Water instead of 11 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 15u1 Master Mix with micropipets of sterile filter tips to each of the Real time PCR reaction plate tubes Separately add 5pl 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 10min Selection of fluorescence channels Target Nucleic Acid Fluorescence measured at 60 C y 10 Threshold setting Choose Ar
3. EU CE Revision No ZJO002 Issue Date Jul 1 2015 Nipah Virus NiV Real Time RT PCR Kit User Manual MBS598102 Instrument I II For use with LightCycler1 0 2 0 Instrument se fre Ws 1 Intended Use Nipah Virus real time RT PCR kit is used for the detection of Nipah Virus in serum plasma infected animal tissue or secretion 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 Nipah virus NiV is an emerging zoonotic virus In infected people Nipah virus causes severe illness characterized by inflammation of the brain encephalitis or respiratory diseases It can also cause severe disease in animals such as pigs resulting in significant economic losses for farmers Nipah vir
4. ent XPCR system without HEX VIC JOE channel may be treated with 111 Molecular Grade Water instead of 1ul IC 1 The volumes of Super Mix and Enzyme Mix per reaction multiply with the number of samples which includes the number of controls 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 20ul Master Mix with micropipets of sterile filter tips to each of the real time PCR reaction plate tubes Separately add Sul RNA sample template positive and negative controls to different 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 10min Icycle 95 C for 15min Icycle 95 C for 15sec 60 C for 1min Fluorescence measured at 60 C Selection of fluorescence channels Target Nucleic Acid HEX VIC JOE AOcycles 5 A 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 and internal co
5. ge 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 Vortex mixer e Cryo container e Sterile filter tips for micro pipets e Disposable gloves powderless e Biohazard waste container e Refrigerator and Freezer e Tube racks e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5ul 10001 e Sterile microtubes 7 A 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 Clinical 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 cooli
6. ithmetic 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 Quality control Negative control positive control and internal control must be performed correctly otherwise the sample results is invalid a ii sepoi M Positive Control qualitative assay 3s 12 Data Analysis and Interpretation The following results are possible Crossing point value z 300m Result Analysis 1 Blank 25 35 Below the detection limit or negative Positive Re test If it is still 35 40 report as 1 Blank PCR Inhibition No diagnosis can be concluded For further questions or problems please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES a IVD Revision No ZJ0004 EU C Issue Date Jul 1 2015 For Research Use Only In USA amp China Nipah Virus NiV Real Time RT PCR Kit 20 C 25 User Manual MBS598102 Instrument I II For use with ABI Prism 7000 7300 7500 7900 Step One Plus iCycler iQ 4 iQ 5 Smart Cycler Il Bio Rad CFX 96 Rotor Gene 6000 Mx3000P 3005P MJ Option2 Chromo4 LightCycler 480 Instrument eo jrm 1 Intended Use Nipah Virus real time RT PCR kit is used for the detection of Nipah Virus in serum plasma infected animal tissue or secretion by using real time PCR systems
7. ld 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 For Research Use Only In USA amp China 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 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 e Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents 9 Procedure 9 1 RNA Extraction RNA extraction kits are available from various manufacturers 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 9 2 Internal Control It is
8. ng 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 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 e Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents 9 Procedure 9 1 RNA Extraction RNA extraction kits are available from various manufacturers 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 1ul rxn and the result will be shown in the HEX VIC JOE 9 3 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 18 1 pl ipl Super Mix Enzyme Mix Internal Control 5yl 20l Extraction RNA Master Mix Reaction Plate Tube l PCR Instrum
9. nsitivity 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 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 a N Warnings and Precaution 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 Clinical samples should be regarded as potentially infectious materials and shou
10. ntrol must be performed HEX VIC JOE correctly otherwise the sample results is invalid AM Molecular Grade Water UNDET 25 35 Positive Control qualitative assay 2 13 Data Analysis and Interpretation The following results are possible Ct value HEX VIC JOE Result Analysis Cess Positives and the software displays the quantitative value For further questions or problems please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
11. tem for the detection of the NiV using 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 NiV RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the NiV 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 NiV 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 4 Kit Contents Type of reagent NiV Super Mix RT PCR Enzyme Mix 1 vial 480u1 1 vial 28 ul 1 vial 400ul 1 vial 30ul 1 vial 30ul Molecular Grade Water Internal Control NiV Positive Control Analysis sensitivity 1 X 10 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 Stora
12. us is closely related to Hendra virus Both are members of the genus Henipavirus a new class of virus in the Paramyxoviridae family Although Nipah virus has caused only a few outbreaks it infects a wide range of animals and causes severe disease and death in people making it a public health concern The Nipah Virus NiV real time RT PCR Kit contains a specific ready to use system for the detection of the NiV using 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 NiV RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the NiV 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 NiV 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 560nm fluorescence of the internal control IC 4 Kit Contents Ref NiV Super Mix 1 vial 350u1 RT PCR Enzyme Mix 1 vial 28ul Molecular Grade Water 1 vial 400u1 Internal Control IC 1 vial 30ul NiV Positive Control 1 vial 30pl Analysis sensitivity 1 X 10 copies ml Note Analysis se
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