G01086 - gerbion
Contents
1. 15 ml reaction tubes Individually packed DNase free RNase free non pyrogenic NukEx TS Shredding material aliquoted in 1 5 or G06007 1 5 2 0 ml safe lock tubes or 2 0 ml screw GO6007 2 0 cap tubes for the manual preparation of samples such as tissue or insects G06007 2 0 sc Proteinase K Proteinase K Molecular Biology Grade 100 mg G07001 gastroplexVirus Instruction Manual Version 1 2 01 04 20152. K2 0 2 ul x N41 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 Put the number of optical PCR reaction tubes needed into the cooling block Pipet 16 ul of the Master Mix into each optical PCR reaction tube Add 4 ul of the eluates from the RNA isolation including the eluate of the water control the Positive Control K3 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 gastroplexVirus Instruction Manual Version 1 2 01 04 2015 1l Table 4 Preparation of the real time RT PCR Master Mix 160 ul Sample 40 ul Total Volume 20 0 ul 16 Instument Settings For the real time RT PCR use the thermal profile shown in Table 5 Table 5 real time RT PCR thermal profile Reverse Transcription 10 min 45 C 1 Initial Denaturation 5 min 95 C 1 Amplification of DNA Denaturation 10 sec 95 G 45 Annealing and Extension 40 sec 60 C Aquisition at the end of this step Dependent on the real time instrument used further instrument settings have to be adjusted according to Table 6 gastroplexVirus Instruction Manual Version 1 2 01 04 2015 12 Table 6 Overview of the instrument settings required for
3. gastroplexVirus Instruction Manual Version 1 2 01 04 2015 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 ethanol free decontaminant Do not combine gastroplexVirus real time RT PCR Kit components of different lot numbers 13 Isolation of Viral Nucleic Acids The gastroplexVirus real time RT PCR is suitable for the detection of Rotavirus Adenovirus and Norovirus in clinical specimens e g stool samples vomit environmental and food samples isolated with suitable isolation methods Commercial kits for the simultaneous isolation of RNA and DNA such as the following are recommended NukEx Pure RNA DNA gerbion Cat No GO5004 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 samples 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
4. PCR If the control of the extraction of nucleic acids is not desired the Control RNA K5 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 gastroplexVirus Instruction Manual Version 1 2 01 04 2015 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 yourself with the real time PCR instrument and read the user manual supplied with the instrument The programm set up In every PCR run one Positive Control K5 and one Negative Control K4 should be included Before each use all reagents except the Enzyme K2 should be thawed completely at room temperature thoroughly 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 ng of the thermal profile should take place before the PCR 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 Protocol A The Control RNA K5 was added during RNA extraction see Control RNA page 8 In this cas
5. ated food or water and by person to person contact For this reason outbreaks of norovirus infection often occur in closed or semi closed communities such as long term care facilities hospitals prisons dormitories and cruise ships Noroviruses are highly contagious and are stable at temperatures between 20 C to 60 C and in acidic environments up to pH 3 Norovirus infections occur throughout the year however in Europe seasonal increases are observed between October and March gastroplexVirus Instruction Manual Version 1 2 01 04 2015 The gastroplexVirus real time RT PCR kit detects Norovirus strains of high genetic diversity such as the following GI Norwalk Desert Shield Winchester Queensarms Southhampton Chiba GII Lordsdale Bristol Melksham Toronto Hawaii Infections with Rotavirus are the n among children Worldwide m infections e die from rotavirus The double strand o d nearly every child infection Rotaviruses are c pathogenic More viruses Adenoviruses mai dependent on the gastroenteritis ke diarrhoea Respira ed RNA ally and infects the enterocytes ehydration seldomly abdominal p respiratory tract occur in correlation immunity develops in the assified than 90 of rotavirus infections are caused by group A nly cau so subsequent infections are less severe By the age of 5 world has nost common cause of se
6. ccured when the samples optical PCR reaction tubes Positive Control reaction tube immedia the same resul components might be work space and ins ely af rumen occurs one or more of contaminated Make sure that in replicates If the result on the contamination were pipetted into the ake sure to pipet the K3 last and close the optical PCR er adding the sample If the kit ts are decontaminated regularly Use a new kit and PCR epeat the real time RT gastroplexVirus Instruction Manual Version 1 2 01 04 2015 19 Other Products 18 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 Description Cat No NukEx Pure RNA DNA Spin column based kit for the isolation GO5004 50 of RNA and DNA from a variety of G05004 200 sample matrices For 50 or 200 extractions NukEx Collection 500 NukEx Collection Tubes for use with GO6008 Tubes ukEx Spin Columns NukEx PLUS 2 0 Reagent for the enzymatic release of GO5016 nucleic acids from swabs and cell culture suspensions Very fast and convenient protocol ncluding NukEx Stop for chemical inactivation NukEx Universal Diluent for samples for real time RT GO1014 Dilution Buffer PCR NukEx Pestle 1 5 ml 100 disposable PBTP pestles for use in GO6006
7. contaminations during RNA extraction will be detectable Please note the chapter Control RNA on page 8 If the real time RT PCR is not performed immediately store the extracted nucleic acids according to the instructions given by the extraction kit s manufacturer Further information about isolation of nucleic acids is to be found in the extraction kit manual or from the extraction kit manufacturers technical service gastroplexVirus Instruction Manual Version 1 2 01 04 2015 14 Control 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 gastroplexVirus Control RNA K5 is added prior to the RNA extraction 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
8. curred 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 gastroplexVirus Instruction Manual Version 1 2 01 04 2015 15 Figure 1 and Figure 2 show examples for positive and negative real time RT PCR results ee f 30000 f i positive sample negative sample e 9 e e ce eec ee aa roe 2 4 6 8 0 2 14 16 19 20 22 24 120 19 30 32 34 36 39 4 4 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 dR P P e d 2 4 0 89 0 12 1 6 1 20 22 24 120 2 30 2 24 36 39 4 42 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 the isolation of the nucleic acids but that the sample is a true negative gastroplexVirus Instruction Manual Version 1 2 01 04 2015 16 18 Troubleshooting 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
9. do not hesitate to contact our scientists on info gerbion com The selected channel for analysis does not comply with the protocol 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 Incorrect configuration of the real time RT PCR Check your work steps and compare with Procedure on page 9 The programming of the thermal profile is incorrect Compare the thermal profile with the protocol Table 5 page 11 Incorrect storage conditions for one or more kit components or kit expired real time RT PCR conditions do not comply with the protocol 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 Iransport and Storage page 3 Check the real time RT PCR conditions page 9 real time RT PCR inhibited Make sure that you use an appropriate isolation method see Isolation of Viral Nucleic Acids page 7 and follow the manufacturer s instructions Make sure that the ethanol containing wash buffer of the isolati
10. e 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 extraction Volume per Reaction Volume Master Mix 15 8 ul Reaction Mix K1 15 8 ul x N 1 0 0 ul Controll RNA K5 0 0 pl x N 1 0 2 ul Enzyme K2 0 2 ul x N 1 gastroplexVirus Instruction Manual Version 1 2 01 04 2015 10 Protocol B The Control RNA K5 is used for the control of the real time RT PCR only see Control RNA page 8 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 pl Control RNA K5 9 ul dH20 before adding it to the Master Mix Table 3 Preparation of the Master Mix Control RNA K5 is added directly to the Master Mix Volume per Reaction Volume Master Mix 15 8 ul Reaction Mix K1 15 8 ul x N 1 0 2 ul Controll RNA K5 diluted 1 10 0 2 ul x N 1 0 2 ul Enzyme
11. gerbo Instruction Manual gastroplexVirus real time RT PCR Kit For the in vitro detection of the RNA of Rotavirus and Norovirus GI and GII and the DNA of Adenovirus in clinical specimens environmental and food samples amp m c IM rmm 18 C E G01086 32 G01086 96 32 96 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 gastroplexVirus Instruction Manual Version 1 2 01 04 2015 Index 1 COrmponelitez oen e Uem n tonat e ndum RR 5 2 AbDreVIatiOfiS cotto tasa ee Mie tet a e detect 5 3 Trarisportiand Stordge see met eo a E EEEE NAE AR 3 4 Intended USE ia citera A a RA INEN coll AE NE INE 3 5 Sample Material octo Re AIRTRAN E CA RR OEE A 4 6 Quality CODO tests tette rte tote te bedeutend 4 7 Product Warranty 8 pigeja iu eio pe 4 9 Priaciple of the Test oett ut dee titan 5 10 Equipment and Reagents to be Supplied by User 6 TI important Noles secet o tete ge eee eT eco tr 6 I2 General PrecalltiOFls ceat triginta lec eetie d ii aie tti erc iret ple 7 55 Js lation OF Viral N eleit AGIS oes htt d Rn 7 T4 Control RNA Kb s tente ro ee tede tei etie 8 I5 Realtime RIEPGR oot ottenere eod Edo ut occa 9 151 Important Points Before Starting ttt 9 152 jieju la gp E 9 l6 Instument Settings eee eee EERE 11 TZ iData AnalysiS
12. irus real time RT PCR is the nucleic acid isolated from clinical specimens e g stool samples vomit environmental or food samples 6 Quality Control In accordance with gerbion s ISO certified Quality Management System each lot of the gastroplexVirus 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 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 Gastroenteritis or infectious diarrhea is an inflammation of the gastrointestinal tract Both the stomach and the small intestine are involved Typical symptoms are diarrhoea vomiting abdominal pain and cramps often followed by dehydration The causative agent can be viral or bacterial Rota Noro and Adenoviruses are the most common viral causes for gastroenteritis Noroviruses are small non enveloped RNA viruses belonging to the family of Caliciviridae They cause approximately 90 96 of epidemic non bacterial outbreaks of gastroenteritis around the world The viruses are transmitted by fecally contamin
13. lification of virus specific fragments are performed in a one step RT PCR The amplification can be detected when specific probes are gastroplexVirus Instruction Manual Version 1 2 01 04 2015 hydrolysed by the Polymerase The emitted fluorescence is measured in the FAM Norovirus ROX Rotavirus and Cy5 Adenovirus channel Furthermore the gastroplexVirus real time RT PCR Kit contains a Control RNA KS 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 TET channel 10 Equipment and Reagents to be Supplied by User e Nucleic Acid isolation kit e g NukEx Pure RNA DNA gerbion Cat No G05004 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 1l Important Notes The gastroplexVirus 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
14. on kit has been completely removed An gastroplexVirus Instruction Manual Version 1 2 01 04 2015 17 additiona centrifugation step at high speed recommended before elution of the RNA Loss of nucleic acids during isolation process In case the Control R extraction the lack of indicate that the nuc an amplification signal eic acid isolation was A KS was added bef ore can not successful Make sure isolation method commercia and stick to the manufacturer hat you use an appropriate kits are recommended s protocol Incorrect storage conditions for one or more components or kit expired 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 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 used a strong fluorescence signal in one detection channel can lead to a weak signal a round 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 preparation of the RT PCR Repeat the real time RT PCR is negative in the repetiti o
15. otavirus RNA In this case detection of a signal of the Control RNA K5 in the VICS HEX JOE V TET channel is inessential as high concentrations of cDNA may reduce or completely inhibit amplification of the Control RNA K5 Asignal in the Cy 5 channel is detected The result is positive the sample contains Adenovirus DNA 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 Norovirus RNA nor Rotavirus RNA nor Adenovirus DNA 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 16 gastroplexVirus Instruction Manual Version 1 2 01 04 2015 14 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 isolation of the nucleic acids was not successful or an inhibition of the RT PCR has oc
16. teet e ce 13 T8 CTEOUDIeSHOOLIDG ic castes eacar tents ea aga Eae eda aat 16 I9 COtherPIOGQUCEo cito eo hte ita aiias tag e Hee Amet 18 gastroplexVirus Instruction Manual Version 1 2 01 04 2015 1 Components The reagents supplied are sufficient for 32 or 96 reactions respectively Table 1 Components of the gastroplexVirus real time RT PCR kit Label Lid Colour Content 32 96 K1 Reaction Mix yellow 1 x506 ul 2x759pu K2 Enzyme blue 1x64ul 1x19 2 ul K3 Positive Control red 1x50 ul 1x1004u Rota Adeno Norovirus K4 Negative Control green 1x50yul 1x1004u K5 Control RNA red 1x160ul 2x 240 u 2 Abbreviations RNA Ribonucleid Acid PCR Polymerase Chain Reaktion RT Reverse Transcription cDNA complementary Deoxyribonucleid Acid 3 Transport and Storage The gastroplexVirus 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 ntended Use The gastroplexVirus real time RT PCR is an assay for the detection of the RNA of Rotavirus and Norovirus and the DNA of Adenovirus in clinical specimens e g stool samples vomit environmental and food samples using real time PCR microplate systems gastroplexVirus Instruction Manual Version 1 2 01 04 2015 5 Sample Material Starting material for the gastroplexV
17. the gastroplexVirus real time RT PCR Norovirus 485 555 i 558 61 LightCycler 4801 Rotavirus peat Control RNA 523 568 Color Compensation Kit Adenovirus 615 670 Multiplex 1 GO70MP1 cc Norovirus 465 510 required i 61 LightCycleragoi Rotavirus 3AF OND Control RNA 498 580 Adenovirus 618 660 Norovirus FAM Gain 8 Stratagene i Mx3000P Rotavirus ROX Gain 1 E Dye Mx3005P Control RNA HEX Gain 1 Adenovirus Cy5 Gain 4 Norovirus FAM ABI 7500 Rotavirus ROX oe Reference Dye ROX Control RNA JOE Adenovirus Cy5 Norovirus Green Rotor Gene Q P Rotor Gene 3000 Rotavirus Orange Rotor Gene 6000 Control RNA Yellow Adenovirus Red gastroplexVirus Instruction Manual Version 1 2 01 04 2015 13 17 Data Analysis The Norovirus specific amplification is measured in the FAM channel the Rotavirus specific amplification in the ROX channel and the Adenovirus 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 Asignal in the FAM channel is detected The result is positive the sample contains Norovirus 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 Asignal in the ROX channel is detected The result is positive the sample contains R
18. vere diarrhoea ore than 450 000 children under 5 years of age ach year Most of them in developing countries virus of the family Reoviridae is transmitted faeca t causes diarrhoea vomiting fever and ain Sometimes infections of the upper with gastroenteritis With each infection at least once gone through a rotavirus into the g se infections of the respiratory system However roups A G among which A C are human serotype numerous other diseases can be caused such as atoconjunctivitis epidemica cystitis rhinitis pharyngitis and nptoms range from mild flu to acute bronchitis and tory syn pneumonia Immu months or years no suppressed patients are prone to severe complications such as acute respiratory distress syndrome Although the epidemiologica characteristics of Adenoviruses vary from type to type all types are transmitted by direct contact feacal orally and rarely by water Some types cause persistent asymptomatic infections of the palatine and pharyngea tonsils and the gastrointestinal tract Spreading of the virus can occur ove 9 Principle of the Test The gastroplexVirus real time RT PCR Kit contains specific primers and hydrolysis probes for the detection of the nucleic acids of Rotavirus Adenovirus and Norovirus in clinical specimens e g stool samples vomit environmental and food samples The reverse transcription RT of viral RNA to cDNA and the subsequent amp
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