Rubella Real TM Qual ENG PCR CE
Contents
1. 40 for clinical samples and the Ct value detected in the FAM Green channel does not exceed the value specified for the Internal Control Ct 35 The fluorescence curve should have a typical sigmoid shape and cross the threshold line once in the region of significant fluorescence increase 2 The sample is considered negative if its Ct in the JOE Yellow HEX channel is not detected the fluorescence curve does not cross the threshold line and the Ct value detected in the FAM Green channel does not exceed the boundary Ct value specified for the Internal Control Ct 35 QUALITY CONTROL PROCEDURE A defined quantity of Internal Control IC is introduced into each sample and control at the beginning of sample preparation procedure in order to control the extraction process of each individual sample and to identify possible reaction inhibition A negative control of extraction NCE positive control of extraction PCE negative amplification control NCA positive amplification control C are required for every run to verify that the specimen preparation the amplification and the detection steps are performed correctly If the controls are out of their expected range see table Results for Controls all of the specimens and controls from that run must be processed beginning from the sample preparation step Sacace Rubella Real TM Qual VER 11 11 2011 PERFORMANCE CHARACTERISTICS Analytical specificity The analyti2. Fl Plate type instruments iQ5 Mx300P ABI 7500 7300 Settings Channel Threshold FAM The threshold line should cross only sigmoid curves of signal accumulation of positive samples and should not cross the baseline HEX Joe C otherwise the threshold level should be raised Set the threshold at y3 a level where fluorescence curves are linear and do not cross curves of the negative samples Sacace Rubella Real TM Qual VER 11 11 2011 SacaceTM Rubella Real TM Qual IVER 11 11 2011 DATA ANALYSIS Accumulation of Rubella virus cDNA amplification product is detected in the JOE Yellow HEX channel Internal Control amplification product is detected in the FAM Green channel The results are interpreted by the software of the PCR instrument used by the crossing or not crossing of the fluorescence curve with the threshold line The results of analysis are considered reliable only if the results obtained for Positive and Negative Controls of Amplification as well as for the Negative Control of Extraction are correct Results for controls Ct in channel Control stage Tor JOE Yellow HE Interpretation control FAM Green x d NCE RNA extraction lt 35 Neg OK PCE RNA extraction lt 35 lt 35 OK NCA RT PCR Neg Neg OK C RT PCR lt 35 lt 35 OK 1 The sample is considered positive if its Ct value detected in the JOE Yellow HEX channel does not exceed the boundary Ct value
3. e PCR for detection of rubella virus RNA in clinical samples TJ Bosma KM Corbett S O Shea Am Soc Microbiol JOURNAL OF CLINICAL MICROBIOLOGY May 1995 p 1075 1079 e An RT PCR assay using oral fluid samples to detect rubella virus genome for epidemiological surveillance A J Vyse L Jin Molecular and Cellular Probes Volume 16 Issue 2 April 2002 Pages 93 97 SacaceTM Rubella Real TM Qual VER 11 11 2011 KEY TO SYMBOLS USED List Number LOT Lot Number lt c For in Vitro Diagnostic Use Store at Manufacturer Consult instructions for use Expiration Date c DE Sacace Rubella Real TM Qual VER C Caution Contains sufficient for lt n gt tests Version Negative Control of Amplification Negative control of Extraction Positive Control of Amplification Internal Control VER 11 11 2011 iQ5 is a registered trademark of Bio Rad Laboratories Rotor Gene Technology is a registered trademark of Qiagen MX3005P is a registered trademark of Agilent Technologies ABIG is a registered trademark of Applied Biosystems LineGeneK is a registered trademark of Bioer SmartCycler is a registered trademark of Cepheid Sacace Biotechnologies Srl mail info gsacace com web www sacace com SacaceTM Rubella Real TM Qual VER via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 4390314892926 11 11 2011
4. 10 min Remove the supernatant leaving 200 ul of the fluid over the pellet Use tips with aerosol barrier Resuspend the pellet Bronchial lavage nasal wash centrifuge at 2000 g min for 10 15 min If the pellet is not visible add 10 ml of liquid and repeat centrifugation Remove and discard the supernatant Resuspend the pellet in 100 ul of Saline water Tissue homogenized with mechanical homogenizer and dissolved in PBS sterile Specimens can be stored at 2 8 C for no longer than 12 hours or frozen at 20 C to 80C Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents RNA ISOLATION The following isolation kits are recommended Ribo Sorb Sacace REF K 2 1 Ribo Virus Sacace REF K 2 C DNA RNA Prep Sacace REF K 2 9 Please carry out the RNA extraction according to the manufacturer s instructions During extraction use the following controls e Positive Control Rubella virus rec Positive Control of Extraction PCE add 90 ul of Negative Control C and 10 ul of RUBELLA RNA C Rec Fag to the tube labeled PCE e Negative Control C add 100 ul of Negative Control C to labeled C e Internal Control STI 87 rec IC add 10 ul of Internal Control RNA during the RNA isolation procedure directly to the sample lysis mixture SacaceTM Rubella Real TM Qual VER 11 11 2011 RE
5. AGENTS PREPARATION REACTION VOLUME 25 pL 1 Prepare required quantity of reaction tubes 2 Prepare for each sample in the new sterile tube Reaction Mix add 10 yl of RT PCR mix 1 5 pl of RT PCR mix 2 0 25 ul of RT G mix 2 0 50 pl of TaqF Polymerase and 0 25 pl of M MLV Revertase Vortex thoroughly and centrifuge for 5 sec This mix must be used immediately Don t store the prepared mix Reagents volume x 1 10 0 5 00 0 25 0 50 0 25 reaction pl N RNA N reactions RT PCR RT PCR RT G TaqF M MLV samples mix 1 mix 2 mix 2 Polymerase Revertase 4 6 60 30 1 5 3 0 1 5 6 8 80 40 2 0 4 0 2 0 8 10 100 50 2 5 5 0 2 5 10 12 120 60 3 0 6 0 3 0 12 14 140 70 3 5 7 0 3 5 58 60 600 300 15 0 30 0 15 0 T specimens plus 2 extraction controls N 2 2 specimens plus extraction and amplification controls N 2 2 3 Add 15 pl of Reaction Mix into each tube 4 Add 10 pl of extracted RNA sample to appropriate tubes with Reaction Mix and mix well by pipetting Re centrifuge all the tubes with extracted RNA for 2 min at maximum speed 12000 16000 g and take carefully supernatant N B don t disturb the pellet sorbent inhibit reaction 5 Prepare for each panel 2 controls e NCA add 10 pl of RT eluent to the tube labeled Negative Control e C add 10 ul of RUBELLA cDNA Pos amp IC Pos to the tube labeled Positive Control The results are interpreted through the presence of crossing of fluore
6. _iSacace BIOTECHNOLOGIES w 1023 For in Vitro Diagnostic Use For Professional Use Only Rubella Real TM Qual Handbook Real Time PCR test for qualitative detection of V REF Sacace Rubella Virus 50 V24 50FRT TM Rubella Real TM Qual VERI 11 11 2011 NAME RUBELLA Real TM Qual INTENDED USE RUBELLA Real TM Qual is a Real Time test for the qualitative detection of Rubella Rosolia Virus RNA in the plasma serum umbilical blood mucosal swabs nasal oral lavages amniotic liquid tissue RUBELLA RNA is extracted from specimens amplified using RT amplification and detected using fluorescent reporter dye probes specific for Rubella or Rubella IC PRINCIPLE OF ASSAY Rubella virus detection by the polymerase chain reaction PCR is based on the amplification of the pathogen genome specific region by using specific primers In real time PCR the amplified product is detected using fluorescent dyes These dyes are linked to oligonucleotide probes that bind specifically to the amplified product The real time monitoring of the fluorescence intensities during the real time PCR allows the detection of accumulating product without re opening of the reaction tubes after the PCR run RUBELLA Real TM Qual PCR kit is a qualitative test that contains the Internal Control IC which must be used in the extraction procedure in order to control the extraction process of each individual sample and to i
7. cal specificity of RUBELLA Real TM Qual PCR kit is ensured by selection of specific primers and probes as well as stringent reaction conditions The primers and probes were checked for possible homologies to all sequences published in gene banks by sequence comparison analysis The clinical specificity of RUBELLA Real TM Qual PCR kit was confirmed in laboratory clinical tests Analytical sensitivity The kit RUBELLA Real TM Qual allows to detect RUBELLA RNA in 10096 of the tests with a sensitivity of not less than 400 copies ml N The claimed analytical features of RUBELLA Real TM Qual PCR kit are guaranteed only when an additional reagent kit DNA RNA prep or RIBO sorb or Ribo Virus is used Target region p150 gene TROUBLESHOOTING The results of analysis are not taken into account in the following cases 1 If the Ct value of a clinical sample detected in the JOE Yellow HEX channel exceeds the boundary Ct value gt 37 the result is considered equivocal It is necessary to repeat the analysis twice If a reproducible positive Ct value is detected the sample is considered to be positive 2 If any Ct value is detected for the Negative Control of Amplification NCA in both channels or the Ct value is detected for Negative Control of Extraction C in the JOE Yellow HEX channel this indicates the contamination of reagents or samples In this case the results of analysis of all samples are considered invalid It is necessary to repeat t
8. dentify possible reaction inhibition RUBELLA Real TM Qual PCR kit uses hot start which greatly reduces the frequency of nonspecifically primed reactions Hot start is guaranteed by separation of nucleotides and Taq polymerase by using a chemically modified polymerase TaqF which is activated by heating at 95 C for 15 min MATERIALS PROVIDED Reagent Description Volume ml Quantity RT G mix 2 colorless clear liquid 0 015 1 tube RT PCR mix 1 FRT Rubella colorless clear liquid 0 6 1 tube RT PCR mix 2 FEP FRT colorless clear liquid 0 3 1 tube Polymerase TaqF colorless clear liquid 0 03 1 tube TM Revertase MMIv colorless clear liquid 0 015 1 tube apis E DNA colorless clear liquid 0 1 1 tube RNA buffer colorless clear liquid 0 6 1 tube Negative Control C straw colored clear liquid 0 5 2 tubes Positive Control Rubella rec colorless clear liquid 0 1 2 tubes deua Pano orice colorless clear liquid 0 5 1 tube must be used in the isolation procedure as Negative Control of Extraction must be used in the isolation procedure as Positive Control of Extraction add 10 ul of RNA C Rec Fag and 90 ul of Negative Control to the tube labeled PCE add 10 ul of Internal Control to each sample during the RNA purification procedure directly to the sample lysis mixture Sacace Rubella Real TM Qual VER 11 11 2011 MATERIALS REQUIRED BUT NOT PROVIDED RNA extraction kit Disposabl
9. e powder free gloves and laboratory coat Pipettes adjustable Sterile pipette tips with aerosol barriers up to 200 ul Tube racks Vortex mixer Desktop centrifuge with a rotor for 2 ml reaction tubes PCR box Personal thermocycler for example RotorGene 6000 Q Qiagen iQ5 Bio Rad Mx3005P Agilent ABI 7500 Applied Disposable polypropylene microtubes for PCR or PCR plate Refrigerator for 2 8 C Deep freezer for s 16 C Waste bin for used tips WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device 1 2 3 Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory O O 9 For In Vitro Diagnostic Use Only Wear disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterward Do not pipette by mouth work areas Do not use a kit after its expiration date Dispose of all specimens and unused reagents in accordance with local regulations Biosafety Level 2 should be used for materials that contain or are suspected of containing infectious agents Clean and disinfect all spills of specimens or reagents using a disinfectant such as 0 596 sodium hypochlorite or other suitable disinfectant Avoid contact of specimens and reagents with the skin eyes and mucous membranes If these solutions come into contact rinse immediately with water and seek medical advice immediately Material Safety Data S
10. h Sacace s ISO 13485 Certified Quality Management System each lot is tested against predetermined specifications to ensure consistent product quality STORAGE INSTRUCTIONS All components of the RUBELLA Real TM Qual PCR kit except for RT G mix 2 RT PCR mix 1 FRT Rubella virus RT PCR mix 2 FEP FRT polymerase TaqF and TM Revertase MMIv are to be stored at 2 8 C All components of the RUBELLA Real TM Qual PCR kit are stable until the expiration date on the label The shelf life of reagents before and after the first use is the same unless otherwise stated RT G mix 2 RT PCR mix 1 FRT Rubella virus RT PCR mix 2 FEP FRT polymerase TaqF and TM Revertase MMIv are to be stored at lt 16 C RT PCR mix 1 FRT Rubella virus is to be kept away from light RUBELLA Real TM Qual PCR kit should be transported at 2 8 for no lon ger than 5 days but should be stored at 2 8 and 20 C immediat ely on receipt STABILITY RUBELLA Real TM Qual Test is stable up to the expiration date indicated on the kit label The product will maintain performance through the control date printed on the label The shelf life of reagents before and after the first use is the same unless otherwise stated Exposure to light heat or humidity may affect the shelf life of some of the kit components and should be avoided Repeated thawing and freezing of these reagents should be avoided as this may reduce the sensitivity Components stored under conditions other tha
11. he analysis of all tests and to take measures to detect and eliminate the source of contamination 3 If the Ct value is absent for the Positive Control of Extraction PCE this indicates improper extraction procedure RNA extraction should be repeated for all samples 4 If the Ct value is absent for the Positive Control of RT PCR C this indicates errors in carrying out PCR or an incorrect amplification program RT PCR should be repeated for all samples 5 If the Ct value of a clinical sample is absent or greater than the boundary Ct value gt 37 for the JOE Yellow HEX channel and the Ct value in the FAM Green channel is greater than the Ct values specified for the Internal Control 237 the result is invalid Analysis of such samples should be repeated starting from the RNA extraction stage SacaceTM Rubella Real TM Qual VER 11 11 2011 REFERENCES e Comparison of four methods using throat swabs to confirm rubella virus infection Zhu Z Xu W Abernathy ES Chen MH Zheng Q Wang T Zhang Z Li C Wang C He W Zhou S Icenogle J J Clin Microbiol 2007 Sep 45 9 2847 52 Epub 2007 Jun 27 e Application of molecular and serological assays to case based investigations of rubella and congenital rubella syndrome Jin L Thomas B J Med Virol 2007 Jul 79 7 101 7 24 e Improved RT PCR for diagnosis and epidemiological surveillance of rubella Cooray S Warrener L Jin L J Clin Virol 2006 Jan 35 1 73 80 Epub 2005 Jul 12
12. heets MSDS are available on request 10 Use of this product should be limited to personnel trained in the techniques of DNA amplification 11 PCR reactions are sensitive to contamination Measures to reduce the risk of contamination in the laboratory include physically separating the activities involved in performing PCR in compliance with good laboratory practice 12 Workflow in the laboratory must proceed in a uni directional manner beginning in the Extraction Area and moving to the Amplification and Detection Area Do not return samples equipment and reagents in the area where you performed previous step Some components of this kit contain sodium azide as a preservative Do not use metal tubing for reagent transfer Sampling of biological materials for PCR analysis transportation and storage N are described in details in the handbook of the manufacturer lt is recommended that this handbook is read before beginning of the work SacaceTM Rubella Real TM Qual VER 11 11 2011 PRODUCT USE LIMITATIONS All reagents may exclusively be used in in vitro diagnostics Use of this product should be limited to personnel trained in the techniques of DNA amplification EN375 Strict compliance with the user manual is required for optimal PCR results Attention should be paid to expiration dates printed on the box and labels of all components Do not use a kit after its expiration date QUALITY CONTROL In accordance wit
13. n those stated on the labels may not perform properly and may adversely affect the assay results SacaceTM Rubella Real TM Qual VER 11 11 2011 SAMPLE COLLECTION STORAGE AND TRANSPORT RUBELLA Real TM Qual can analyze RNA extracted from Peripheral and umbilical cord blood plasma Collect blood to a Vacuett tube lavender cap 6 EDTA after overnight fasting or at least 3 h after the patient had a meal Invert the tube several times to ensure proper mixing of blood with the anticoagulant Centrifuge the tube with blood at 800 1600 g at room temperature for 20 min Take 1 0 ml of plasma and transfer it to a sterile 2 0 ml Eppendorf tube Saliva Collect 0 2 1 0 ml of saliva to a 1 5 ml Eppendorf tube Have the patient to rinse his mouth with water 3 times before sampling saliva Oropharyngeal swabs are obtained with a dry cotton probe from the tonsillar area palatine arches and posterior oropharyngeal surface Have a patient to rinse his mouth with water before swabbing After sampling the cotton end of the probe should be placed into a sterile tube containing 500 yl of transport medium Then the probe should be broken off at the score mark and the tube should be tightly closed Amniotic fluid should be obtained during amniocentesis by the standard procedure and collected to a sterile Eppendorf tube Thoroughly resuspend the obtained sample and transfer 1 ml of it to a new sterile tube Centrifuge the tube at 8 000 9 000 g for
14. scence curve with the threshold line Rubella cDNA is detected on the JOE Yellow HEX Cy3 channel IC DNA on the FAM Green channel Sacace Rubella Real TM Qual VER 11 11 2011 AMPLIFICATION Program the real time instrument according to manufacturer s manual Amplification program for rotor t pe instruments Step _ Temperature C Fluorescence detection Hed 5 mn Cycling2 6 20s Ass JOEVeIow JOE Yellow 7a Be j o o Fluorescence is detected at the 2nd step of Cycling 2 stage 60 in FAM Green and JOE Yellow fluorescence channels Amplification program for plate and modular type instruments Step Temperature C Time Fluorescence detection Repeats 4 e 3039 ism ee e fs 2 9 j mn AJ 21 3 60 C 20s pe L e S o Pc 5s ooo d Fluorescence i is detected at stage 4 60 C in FAM and HEX fluorescence channels For example Rotor Gene 6000 Q Corbett Research Qiagen 2 For example iQ5 BioRad Mx3005P Stratagene Applied Biosystems amp 7500 Real Time PCR Applera SmartCycler amp Cepheid Rotor type instruments Rotor Gene 3000 6000 Rotor Gene Q etc Settings Sus aa More Settings Slope Channel Optimisation Threshold Outlier cons Dynamic tube P Removal FAM Gre from 3 Flto 8 Ae Fl 0 03 10 96 On On JOE Yell from 3 FI to 8 0 03 10 On On OW
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