G01084 - gerbion
Contents
1. g erb on Instruction Manual respiraRNA 2 0 real time RT PCR Kit For the in vitro detection of the RNA of Influenza A virus Influenza B virus and Respiratory Syncytial Virus in clinical specimens A TH TI G01084 32 G01084 96 m 52 96 18 C gerbion gmbH amp Co KG Remsstr 1 70806 Kornwestheim Germany phone 49 7154 806 20 0 fax 49 7154 806 20 29 e mail info gerbion com www gerbion com E respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 Index 1 COMO ONEN rennen a a a a aa a aaas 3 2 A ee 3 5 Mans DO ana Sterage rt A seine 3 4 mended US Sa a ee essen 5 5 A PRT RO eR RW 4 6 OU SEO A een ined cata sts eta cant eaten te ata 4 74 A E EEEE EE AEA 4 8 Talis to Bch lle ae a E RCRRULSTEIT TE te SUT Oe eI 4 9 PERIEID ESOT U EN 5 10 Equipment and Reagents to be Supplied by Use aia 6 F OA E O 6 12 General Pie a ONS een E EE sense 6 ES ASNO la 7 iA Contool RNAAK aida 7 13 Re LUDER RER en ee 8 Es all important Points Before Stalin oa 8 15 2 A a ee 8 E Data Anal vis see I re TE PRED 14 I TOUDE Noon satten OA PEI ten nates 17 18 Qer POUC ogis ea r a 19 respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 1 Components The reagents supplied are sufficient for 52 or 96 reactions respectively Table 1 Components of the respiraRNA real time RT PCR kit Lid Colour Content 32 96 Reaction Mix 1 x 506 ul 2X799 ul Enzyme 1x64 ul Lx 192 ul Positive Control Le SON 1x 100 u2. Mx3005P ABI 7500 Rotor Gene Q Rotor Gene 3000 Rotor Gene 6000 Parameter Influenza A Virus RSV Control RNA Influenza B Virus Influenza A Virus RSV Control RNA Influenza B Virus Influenza A Virus RSV Control RNA Influenza B Virus Influenza A Virus RSV Control RNA Influenza B Virus Influenza A Virus RSV Control RNA Influenza B Virus Detection Channel 485 555 558 610 525 568 615 670 465 510 555 610 498 580 618 660 Green Orange Yellow Red Notes Color Compensation kit Multiplex 1 GO7OMP1 cc required Gain 8 Gain 1 Gain 1 Gain 4 Reference Dye None Option Reference Dye ROX NO respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 14 16 Data Analysis The Influenza A virus specific amplification is measured in the FAM channel the Respiratory Syncytial Virus specific amplification in the ROX channel and the Influenza B virus specific amplification in the Cy 5 channel The amplification of the Control RNA K5 is measured in the VIC HEX JOE TET channel Following results can occur e A signal in the FAM channel is detected The result is positive the sample contains Influenza A virus RNA In this case detection of a signal of the Control RNA K5 in the VIC HEX JOE TET channel is inessential as high concentrations of cDNA may reduce or completely inhibit amplification of the Control RNA K5 A signal in the ROX channel is detected The result is p
3. TET channel 10 Equipment and Reagents to be Supplied by User RNA isolation kit e g NukEx Pure RNA DNA gerbion Cat No GO5004 o NukEx PLUS 2 0 Nucleic Acid Release Reagent gerbion Cat No GO5016 Sterile microtubes Pipets adjustable volume Sterile pipet tips with filter Table centrifuge Vortexer Real time PCR instrument Optical PCR reaction tubes with lid Optional Liquid handling system for automation 11 Important Notes e The respiraRNA 2 0 real time RT PCR must be performed by qualified personnel only Good Laboratory Practice GLP has to be applied Clinical samples must always be regarded as potentially infectious material and all equipment used has to be treated as potentially contaminated 12 General Precautions Stick to the protocol described in the Instruction Manual Set up different laboratory areas for the preparation of samples and for the set up of the RT PCR in order to avoid contaminations Pipettes tubes and other materials must not circulate between those different laboratory areas The Enzyme K2 is liquid even at 18 C Take it out of the freezer shortly before usage and put it back immediately Always use filter tips Regulary decontaminate equipment and benches with ethanohol free decontaminant respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 Do not combine respiraRNA 2 0 real time RT PCR Kit components of different lot numbers 13 Isolation of Viral RNA The respiraRNA 2 0 real time R
4. A isolation was not successful or an inhibition of the RT PCR has occurred In case the Control RNA K5 was added during RNA isolation and not directly to the PCR Master Mix the Negative Control K4 is negative in both channels respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 16 Figure 1 and Figure 2 show examples for positive and negative real time RT PCR results 2 4 6 8 10 12 14 6 18 20 22 24 26 26 30 2 N 3506 38 WO 2 4 Cycles Figure 1 The positive sample shows virus specific amplification in the FAM channel whereas no fluorescence signal is detected in the negative sample Fluorescence GR 2 4 6 S 0 2 4 6 8 20 22 24 20 28 320 32 N BH BW ax 2 4 Cycles Figure 2 The positive sample as well as the negative sample show a signal in the Control RNA specific VIC HEX JOE TET channel The amplification signal of the Control RNA K5 in the negative sample shows that the missing signal in the virus specific FAM channel is not due to RT PCR inhibition or failure of RNA isolation but that the sample is a true negative respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 17 Troubleshooting 17 The following troubleshooting guide is included to help you with possible problems that may arise when performing a real time RT PCR If you have further questions please do not hesitate to contact our scientists on info gerbion com No fluorescence signal in the FAM ROX Cy 5 channel of the Positive C
5. T PCR is suitable for the detection of Influenza A virus RSV Influenza B virus in clinical specimens e g throat swabs nasal swabs bronchoalveolar lavage liquor isolated with suitable isolation methods Commercial kits for RNA isolation such as the following are recommended NukEx Pure RNA DNA gerbion Cat No G05004 Alternatively RNA can be released from throat or nasal swabs with NukEx PLUS 2 0 Nucleic Acid Release Reagent gerbion Cat No GO5016 This is the fastest and most convenient method for the release of nucleic acid from swabs because column based purification of the RNA can be omitted More information can be found on www gerbion com Important In addition to the samples always run a water control in your extraction Treat this water control analogous to a sample Comparing the amplification of the Control RNA K5 in the sample to the amplification of the internal control in the water control will give insights on possible inhibitions of the real time RT PCR Furthermore possible contaminations during RNA extraction will be detectable Please note the chapter Control RNA on page 7 If the real time RT PCR is not performed immediately store extracted RNA and NukEx PLUS 2 0 lysates according to the instructions given by the RNA extraction kit s manufacturer Further information about RNA isolation is to be found in the extraction kit manual or from the extraction kit manufacturer s technical service 14 Co
6. The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse gerbion will replace it free of charge or refund the price We reserve the right to change alter or modify any product to enhance its performance and design 8 Introduction respiraRNA 2 0 is a multiplex real time RT PCR for the detection of causative agents of respiratory diseases The respiraRNA 2 0 real time RT PCR is designated for pathogens with a RNA genome Influenza virus A Influenza virus B RSV In combination with NukEx PLUS 2 0 gerbion Cat No GO5016 for the preparation of throat swabs and nasal swabs respiraRNA 2 0 real time RT PCR allows for fast efficient and cost effective diagnostics Influenza viruses belong to the family of Orthomyxoviridae and are the causative agent of the flu Influenza A and B viruses have a single stranded RNA genome consisting of 8 RNA segments The genome of Influenza A viruses is characterized by a high mutation frequency the so called antigenic drift Numerous subtypes of Influenza A viruses are known They can be catergorized by their surface antigenes H haemagglutinin and N neuraminidase Influenza A H1N1 Virus Influenza A H5N1 Virus etc There are three ways of transmission of Influenza viruses respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 droplet infection through inhalation of contaminat
7. each optical PCR reaction tube Add 4 ul of the eluates from the RNA isolation including the eluate of the water control or inactivated crude NukEx PLUS lysates the Positive Controls K3 K4 KS and the Negative Control K4 to the corresponding optical PCR reaction tube Table 4 Close the optical PCR reaction tubes immediately after filling in order to reduce the risk of contamination Table 4 Preparation of the real time PCR Component Volume Master Mix Sample Total Volume respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 12 For the real time RT PCR use the thermal profile shown in Table 5 Table 5 real time RT PCR thermal profile Discription Temperature Number of Cycles Reverse Transcription 45 C 1 Initial Denaturation 95 C 1 Amplification of cDNA Denaturation 10 sec 95 C Annealing and Extension AO sec 60 C Aquisition at the end of this step Important Crude NukEx PLUS 2 0 lysates must be inactivated prior to adding them to the real time PCR mix when performing a reverse transcriptase step prior to the initial denaturation Dependent on the real time instrument used further instrument settings have to be adjusted according to Table 6 respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 13 Table 6 Overview of the instrument settings required for the respiraDNA real time PCR Real time RT PCR Insttrument LightCycler 4801 LightCycler 480I Stratagene Mx3000P
8. ed aerosols in the air contact infection through direct contact with infected persons smear infection through contaminated surfaces e g door handle telephone etc According to the World Health Organisation s WHO estimates 10 to 20 of the world s population are affected each year 80 of all infections with Influenza virus remain unnoticed or proceed as a mild cold while the remaining 20 lead to severe disease Symptoms usually occur suddenly At first chills and general feeling of illness with fever up to 40 C occur In the further course of the disease following symptoms are common sore throat dry cough flu nausea headache and limb pains Most of the fatalities in connection with Influenza are not due to the primary virus infection but due to bacterial infections These secondary infections lead to pneumonia ear infection and myocarditis Respiratory Syncytial Viruses are enveloped negative sense single stranded RNA Viruses of the Paramyxoviridae familiy RSV is a member of the subfamily Pneumovirinae genus Pneumovirus Human Respiratory Syncytial Viruses are divided into subgroups A and B Transmission of the virus mainly takes place by smear and droplet infection Typical symptoms are flu acute bronchiolitis and otitis media Infants suffer from fever coughing and breathing difficulties 5 of the affected children develop pseudo croup Further SBV infections are considered to be a risk factor for sudden infant death s
9. gent for the enzymatic release of nucleic acids from swabs and cell culture suspensions Very fast and convinient protocol Including NukEx Stop for chemical inactivation 100 disposable PBTP pestles for use in 1 5 mi reaction tubes Individually packed DNase free RNase free non pyrogenic Shredding material aliquoted in 1 5 or 2 0 ml safe lock tubes or 2 0 ml screw cap tubes for the manual preparation of samples such as tissue or insects Proteinase K Molecular Biology Grade from Tritirachium album 100 mg Cat No GO5004 50 605004 200 06008 GO5016 G06006 606007 1 5 606007 2 0 606007 2 0 sc 607001 respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015
10. ion Volume per Reaction Volume Master Mix 15 8 ul Reaction Mix K1 15 8 ul x N 1 0 0 ul Control RNA K5 0 0 ul x N 1 0 2 ul Enzyme K2 0 2 ul x N 1 Protocol B The Control RNA K5 is used for the control of the real time RT PCR only see Control RNA page 7 In this case prepare the Master Mix on ice or in a cooling block 2 to 8 C according to Table 3 The Master Mix contains all of the components needed for real RT PCR except the sample Prepare a volume of Master Mix for at least one sample more than required in order to compensate for pipetting inaccuracy Important Dilute the Control RNA K5 1 10 in sterile dH O e g 1 ul Control RNA K5 9 ul dH O before adding it to the Master Mix respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 11 Table 3 Preparation of the Master Mix Control RNA K5 is added directly to the Master Mix 15 8 ul Reaction Mix K1 15 8 ul x N 1 0 2 ul Control RNA K5 diluted 1 10 0 2 ul x N 1 0 2 ul Enzyme K2 0 2 ul x N 1 The increase in volume caused by adding the Control RNA is not taken into account when preparing the PCR assay The sensitivity of the detection system is not impaired Protocol A and B real time RT PCR set up Important Crude NukEx PLUS 2 0 lysates must be inactivated prior to adding them to the real time PCR mix Put the number of optical PCR reaction tubes needed into the cooling block Pipet 16 ul of the Master Mix into
11. l Influenza A Influena B RSV Negative Control 1x 50 ul 1x 100 ul Control RNA 1x160ul 2 x 240 ul 2 Abbreviations RNA Ribonucleid Acid PCR Polymerase Chain Reaction RT Reverse Transcription cDNA complementary Deoxyribonucleid Acid RSV Respiratory Syncytial Virus 3 Transport and Storage The respiraRNA 2 0 real time RT PCR Kit is shipped on dry ice All components must be stored at 18 C in the dark immediately after receipt Do not use reagents after the date of expiry printed on the package After initial usage reagents are stable for up to six months 4 Intended Use The respiraRNA 2 0 real time RT PCR is an assay for the detection of RNA of Influenza A virus RSV Influenza B virus in clinical specimens e g throat swabs nasal swabs bronchoalveolar lavage liquor using real time PCR microplate systems respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 5 Sample Material Starting material for the respiraRNA 2 0 real time RT PCR is viral RNA isolated from clinical specimens e g throat swabs nasal swabs bronchoalveolar lavage liquor 6 Quality Control In accordance with gerbion s ISO certified Quality Management System each lot of the respiraRNA 2 0 real time RT PCR Kit is tested against predetermined specifications to ensure consistent product quality 7 Product Warranty gerbion guarantees the performance of all products when used according to the instructions given in the Instruction Manual
12. ntrol RNA K5 A Control RNA K5 is supplied to be used as Extraction Control This allows the user to control the RNA isolation procedure and to check for possible real time RT PCR inhibition Control RNA K5 used as Extraction Control respiraRNA 2 0 Control RNA K5 is added prior to the RNA extraction respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 To this end multiply the buffer volume needed per extraction with the number of samples including at least one water control N plus 1 to compensate for inaccuracies in pipetting N 1 Add 5 ul Control RNA K5 per extraction 5 ul x N 1 Mix well Perform the RNA isolation according to the manufacturer s instructions If the extraction protocol includes an incubation step of the sample in the first buffer the Control RNA K5 is to be added to each sample individually after incubation The Control RNA K5 must not be added to the sample material directly Control RNA K5 used as Internal Control of the real time RT PCR If crude NukEx PLUS 2 0 lysates are being used or control of the RNA extraction is not desired the Control RNA KS can be used as Internal Control of the real time RT PCR only To that end the Control RNA K5 is to be added directly to the real time RT PCR Master Mix 15 Real time RT PCR 15 1 Important Points Before Starting Please pay attention to the Important Notes on page 6 Before setting up the real time RT PCR familiarise you
13. ontrols K3 K4 K5 The selected channel for analysis does not comply with the protocol Incorrect configuration of the real time RT PCR The programming of the thermal profile is incorrect Incorrect storage conditions for one or more kit components or kit expired Select the FAM channel for analysis of the Norovirus specific amplification the ROX channel for analysis of the Rotavirus specific amplification the Cy 5 channel for analysis of the Adenovirus specific amplification and the VIC HEX JOETM TET channel for the amplification of the Control RNA K5 Due to amplification in all three specific channels amplification of the Internal Control can be inhibited in the Positive Control K3 Check your work steps and compare with Procedure on page 8 Compare the thermal profile with the protocol Table 5 page 12 Check the storage conditions and the date of expiry printed on the kit label If necessary use a new kit and make sure kit components are stored as described in Transport and Storage page 3 Weak or no signal of the Control RNA K5 and simultaneous absence of a signal in the virus specific FAM channel ROX channel or Cy 5 channel real time RT PCR conditions do not comply with the protocol real time RT PCR inhibited Check the real time RT PCR conditions page 8 Make sure that you use an appropriate isolation method see Isolation of Viral RNA page 7 and follow the manufacturer s in
14. ositive the sample contains Respiratory Syncytial Virus RNA In this case detection of a signal of the Control RNA K5 in the VIC HEX JOE TET channel is inessential as high concentrations of cDNA may reduce or completely inhibit amplification of the Control RNA K5 e A signal in the Cy 5 channel is detected The result is positive the sample contains Influenza B virus RNA In this case detection of a signal of the Control RNA K5 in the VIC HEX JOE TET channel is inessential as high concentrations of cDNA may reduce or completely inhibit amplification of the Control RNA K5 No signal in the FAM ROX and Cy5 channel but a signal in the VIC HEX JOE TET channel is detected The result is negative the sample does neither contain Influenza A virus RNA nor Respiratory Syncytial Virus RNA nor Influenza B virus RNA The signal of the Control RNA K5 excludes the possibilities of RNA isolation failure in case the Control RNA K5 is being used as an Extraction Control and or real time RT PCR inhibition If the C value of a sample differs significantly from the C value of the water control a partial inhibition occured which can lead to negative results in weak positive samples see Troubleshooting page 17 respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 15 Neither in the FAM ROX Cy 5 nor in the VIC HEX JOE TET channel a Signal is detected A diagnostic statement cannot be made The RN
15. rself with the real time PCR instrument and read the user manual supplied with the instrument The programming of the thermal profile should take place before the PCR set up In every PCR run a Positive Control K3 and one Negative Control K4 should be included Before each use all reagents except the Enzyme K2 should be thawed completely at room temperature thouroughly mixed do NOT vortex the Reaction Mix K1 but mix by pipetting up and down repeatedly and centrifuged very briefly Then place all reagents on ice or on a cooling block 2 to 8 C 15 2 Procedure If the Control RNA K5 is used to control both the real time RT PCR and the RNA isolation procedure please follow protocol A If the Control RNA K5 is solely used to detect possible inhibition failure of the real time RT PCR please follow protocol B respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 10 Protocol A The Control RNA K5 was added during RNA extraction see Control RNA page 7 In this case prepare the Master Mix on ice or in a cooling block 2 to 8 C according to Table 2 The Master Mix contains all of the components needed for RT PCR except the sample Prepare a volume of Master Mix for at least one sample more than required in order to compensate for pipetting inaccuracy Table 2 Preparation of the Master Mix Control RNA K5 was added during RNA extract
16. sed a strong fluorescence signal in one detection channel can lead to a weak signal around CT 40 in another channel due to so called cross talk between channels Detection of a fluorescence signal in the FAM channel ROX channel or Cy 5 channel of the Negative Control K4 Contamination during Repeat the real time RT PCR in replicates If the result preparation of the RT PCR is negative in the repetition the contamination occured when the samples were pipetted into the optical PCR reaction tubes Make sure to pipet the Positive Control K3 last and close the optical PCR reaction tube immediately after adding the sample If the same result occurs one or more of the kit components might be contaminated Make sure that work space and instruments are decontaminated regularly Use a new kit and repeat the real time RT PCR respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 18 Other Products 19 A number of products related to real time PCR and nucleic acid isolation is available from gerbion GmbH amp Co KG More information as well as the complete Product Catalogue is available on www gerbion com Product NukEx Pure RNA DNA NukEx Collection Tubes NukEx PLUS 2 0 NukEx Pestle 1 5 ml NukEx TS Proteinase K Description Spin column based kit for the isolation of RNA and DNA from a variety of sample matrices For 50 or 200 extractions 500 NukEx Collection Tubes for use with NukEx Spin Columns Rea
17. structions Make sure that the ethanol containing washing buffer of the isolation kit has been completely removed An additional centrifugation step at high speed is recommended before elution of the RNA Dilute NukEx PLUS 2 0 lysates 1 3 in H gt 0 or NukEx Universal Dilution Buffer gerbion Cat No GO1014 Alternatively purify the respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 18 lysates with e g NukEx Pure RNA DNA Kit gerbion Cat No GO5004 RNA loss during isolation In case the Control RNA K5 was added before process extraction the lack of an amplification signal can indicate that the RNA isolation was not successful Make sure that you use an appropriate isolation method commercial kits are recommended and stick to the manufacturer s protocol NukEx PLUS 2 0 crude Crude NukEx PLUS lysates must be inactivated prior to lysates not inactivated adding them to the real time RT PCR mix when performing a reverse transcriptase step prior to the initial denaturation Incorrect storage conditions Check the storage conditions and the date of expiry for one or more printed on the kit label If necessary use a new kit and components or kit expired make sure kit components are stored as described in Transport and Storage page 3 Detection of a weak fluorescence signal in the FAM channel of a sample with a strong fluorescence signal in the Cy5 channel Cross talk Depending on the real time PCR instrument u
18. yndrome SIDS 40 7 of all children undergo RSV infection under the age of 1 Nearly every child has once been infected by the end of the second year of their lives Infection with RSV does not lead to immunity re infections can occur throughout life causing mild symptoms in healthy persons 9 Principle of the Test The respiraRNA 2 0 real time RT PCR Kit contains specific primers and hydrolysis probes for the detection of the RNA of Influenza A virus RSV and Influenza B virus in clinical specimens e g throat swabs nasal swabs bronchoalveolar lavage liquor after the extraction of RNA from the sample material The reverse transcription RT of viral RNA to cDNA and the subsequent amplification of virus specific fragments are performed in a one step RT PCR The amplification can be detected when specific probes are hydrolysed by the Polymerase The emitted fluorescence is measured in the FAM Influenza A virus ROX RSV and Cy5 nfluenza B virus channel respiraRNA 2 0 Instruction Manual Version 2 1 05 05 2015 Furthermore the respiraRNA 2 0 real time PCR Kit contains a Control RNA K5 which is detected in a second amplification system Added during RNA extraction the Control RNA K5 allows not only for the detection of RT PCR inhibition but also detects possible mistakes during RNA extraction This greatly reduces the risk of false negative results The fluorescence of the Control RNA K5 is measured in the VIC HEX JOE
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