NHS Meningitidis Real TM ENG PCR ver 21032013 - bio
Contents
1. H influenzae e add 10 ul of Pos DNA H influenzae C to the tube with PCR mix 1 S pneumoniae H influenzae e add 10 ul of Pos DNA N meningitidis C to the tube with PCR mix 1 N meningitidis IC e add 10 ul of Pos IC C to the tube with PCR mix 1 N meningitidis IC Amplification Create a temperature profile on your Real time instrument as follows Rotor type instruments Plate type or modular instruments Fluorescence Cycle 5 Fluorescence Cycle SER vema e vine detection repeats Tema E Vo detection repeats Hold 95 15 min 1 95 15 min 1 95 10s 95 10s B FAM Green 30s FAM Cycling 56 208 JOE Yellow 45 9e JOE HEX Cy3 45 72 10s 72 10s For example Rotor Gene 3000 6000 Q Corbett Research Qiagen 2 For example SaCycler 96 Sacace CFX iQ5 BioRad Mx3005P Agilent ABIO 7300 7500 StepOne Real Time PCR Applied Biosystems SmartCycler Cepheid LineGeneK Bioer Sacace NHS Meningitis Real TM VER 21 03 2013 INSTRUMENT SETTINGS Rotor type instruments Calibrate Gain More Settings eelo Optimisation Manek Outlier Removal Aopo Sensa FAM Green from 5 Fl to 10 Fl 0 05 10 On JOE Yellow from 4 FI to 8 Fl 0 05 1096 On Plate type instruments The threshold line should cross only sigmoid curves of signal accumulation of positive samples and should not cross the baseline otherwise the threshold2. e Contamination during PCR preparation procedure All samples results are invalid Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol or special DNA decontamination reagents Pipette the Positive controls at the end Repeat the PCR preparation with the new set of reagents Sacace NHS Meningitis Real TM VER 21 03 2013 Sacace NHS Meningitis Real TM VER 21 03 2013 KEY TO SYMBOLS USED List Number LOT Lot Number For in Vitro Diagnostic Use Store at Manufacturer Consult instructions for use Expiration Date ro E SaCyclerT is a registered trademark of Sacace Biotechnologies CFX and iQ5 are registered trademarks of Bio Rad Laboratories Rotor Gene is a registered trademark of Qiagen MX3005P is a registered trademark of Agilent Technologies ABIO is a registered trademark of Applied Biosystems LineGeneK is a registered trademark of Bioer SmartCycler is a registered trademark of Cepheid Sacace Biotechnologies Srl VER NCA NCE Caution Contains sufficient for n tests Version Negative Control of Amplification Negative control of Extraction Positive Control of Amplification Internal Control via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 4390314892926 mail info sacace com web www sacace com Sacace NHS Meningitis Real TM VER 21 03 2013
3. 0 mi e Pos DNAN meningitidis C 0 1 ml e Pos DNA H influenzae C 0 1 ml e Pos DNA S pneumoniae C 0 1 ml e Pos IC C 0 1 ml e DNA buffer 0 5 ml Part N 3 NHS Meningitis Real TM RealTime amplification e PCR mix 1 N meningitidis IC 0 6 ml e PCR mix 1 S pneumoniae H influenzae 0 6 ml e PCR mix 2 2 x 0 3 ml e TaqF Polymerase 2 x 0 03 ml Contains reagents for 55 reactions E must be used in the isolation procedure as Negative Control of Extraction i add 10 ul of Internal Control during the DNA purification procedure directly to the sample lysis mixture Sacace NHS Meningitis Real TM VER 21 03 2013 MATERIALS REQUIRED BUT NOT PROVIDED Zone 1 sample preparation e DNA extraction kit Module No 1 e Biological cabinet e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g Eppendorf 5415D or equivalent e 60 C 29C dry heat block e Vortex mixer e Pipettors capacity 5 40 ul 40 200 ul 200 1000 ul with aerosol barrier e Sterile pipette tips with filters e 1 5 ml polypropylene sterile tubes Sarstedt ASP Eppendorf e Disposable gloves powderless e Biohazard waste container e Refrigerator Freezer Zone 2 RT and amplification e Real Time Thermal cycler e Reaction tubes e Workstation e Pipettes adjustable e Sterile pipette tips with filters e Vortex mixer e Freezer refrigerator Sacace NHS Meningitis Real TM VER 21 03 2013 STORAGE INST
4. 3 Cerebrospinal fluid Prep Real TM densae 1x10 Streptococcus pneumoniae Genome equivalents GE of the microorganism per 1 ml of a clinical sample Specificity The analytical specificity of NHS Meningitis Real TM PCR kit is ensured by selection of specific primers and probes as well as strict reaction conditions The primers and probes were checked for possible homologies to all sequences deposited in gene banks by sequence comparison analysis Specificity was evaluated by testing the following microorganism sand strains Enterobacter aerogenes and E cloacae Enterococcus faecalis GISK 29212 Escherichia coli NCTC 9001 and E coli ATCC 25922 Haemophilus parainfluenzae and H haemolyticus Klebsiella oxytoca and K pneumoniae Listeria monocytogenes Moraxella catarrhalis Neisseria cinereae N elongate N flavescens N gonorrhoeae N mucosa N sicca and N subflava Pantoea agglomerans Proteus mirabilis Pseudomonas aeruginosa ATCC 27853 Salmonella enteritidis GISK 1137 and S typhi Central Public Health Laboratory London 5715 Shigella flexneri 2a GISK 1270 and S sonnei GISK 9090 Staphylococcus aureus ATCC 25923 and S saprophyticus ATCC 15305 S pneumoniae S agalactiae S milleri S mitis S mutans S pyogenes S salivarius S sanguis S suis and S viridians and Yersinia enterocolitica and Y pseudotuberculosis The analytical specificity was also confirmed by testing human DNA Non specific resu
5. all tubes with open cap for 5 min at 65 C Resuspend the pellet in 50 ul of DNA eluent Incubate for 5 min at 65 C and vortex periodically Centrifuge the tubes for 2 min at maximum speed 12000 16000 g The supernatant contains DNA ready for amplification The amplification can be performed on the same day of extraction Sacace NHS Meningitis Real TM VER 21 03 2013 PROTOCOL Total reaction volume is 25 ul the volume of DNA sample is 10 ul 1 Prepare required quantity of reaction tubes 2 tubes for each sample Controls 2 Prepare the reaction mix for required number of samples 3 For N reactions mix for each PCR Mix 1 in a new tube 10 N 1 ul of PCR mix 1 S pneumoniae H influenzae or N meningitidis lC 5 0 N 1 pl of PCR mix 2 0 5 N 1 pl of TaqF Polymerase 4 Vortex the tube then centrifuge shortly Add 15 pl of prepared reaction mix into each appropriate tube 5 Using tips with aerosol filter add 10 pl of DNA samples obtained at the stage of DNA isolation and mix carefully by pipetting N B If the DNA Sorb isolation kit is used as a DNA extraction kit re centrifuge all the tubes with extracted DNA for 2 min at maximum speed 12000 16000 g and take carefully supernatant N B don t disturb the pellet sorbent inhibit reaction 6 Prepare for each panel 3 controls e add 10 ul of DNA buffer to the tube labeled Amplification Negative Control e add 10 pl of Pos DNA S pneumoniae C to the tube with PCR mix 1 S pneumoniae
6. ION 1 ar wo N o PINO 10 11 12 13 16 17 Lysis Solution and Washing Solution in case of their storage at 2 8 C should be warmed up to 60 C until disappearance of ice crystals Prepare required quantity of 1 5 ml polypropylene tubes Add to each tube 300 ul of Lysis Solution and 10 ul of IC Add 100 yul of Samples to the appropriate tube Prepare Controls as follows e add 100 ul of C Negative Control to labeled Cneg Vortex the tubes incubate 5 min at 65 C and centrifuge for 5 sec Vortex vigorously Sorbent and add 25 ul to each tube Vortex for 5 7 sec and incubate all tubes for 10 min at room temperature Vortex periodically Centrifuge all tubes for 1 min at 5000g and using a micropipette with a plugged aerosol barrier tip carefully remove and discard supernatant from each tube without disturbing the pellet Change tips between tubes Add 300 ul of Washing Solution 1 to each tube Vortex vigorously and centrifuge for 1 min at 5000g and using a micropipette with a plugged aerosol barrier tip carefully remove and discard supernatant from each tube without disturbing the pellet Change tips between tubes Add 500 ul of Washing Solution 2 to each tube Vortex vigorously and centrifuge for 1 min at 10000g and using a micropipette with a plugged aerosol barrier tip carefully remove and discard supernatant from each tube without disturbing the pellet Change tips between tubes Repeat step 11 Incubate
7. RUCTIONS Part N 1 DNA Sorb B must be stored at 2 8 C Part N 2 Controls must be stored at 2 8 C Part N 3 NHS Meningitis Real TM must be stored at 20 C The kit can be shipped at 2 8 C but should be stored at 2 8 C and 20 C immediately on receipt STABILITY NHS Meningitis Real TM 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 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 QUALITY CONTROL In accordance with Sacace s ISO 13485 Certified Quality Management System each lot is tested against predetermined specifications to ensure consistent product quality Sacace NHS Meningitis Real TM VER 21 03 2013 WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device For In Vitro Diagnostic Use Only The user should always pay attention to the following ZN Lysis Solution contains guanidine thiocyanate Guanidine thiocyanate is harmful if inhaled or comes into contact with skin or if swallowed Contact with acid releases toxic gas Xn R 20 21 22 36 37 38 S 36 37 39 e Use sterile pipette tips with aerosol barriers and use new tip for every procedure e Store extracted positive material samples controls and ampli
8. _iSacace BIOTECHNOLOGIES For in Vitro Diagnostic Use NHS Meningitis Real TM Handbook Real time PCR kit for detection of Neisseria meningitidis Haemophilus influenzae and Streptococcus pneumoniae REF B25 50FRT REF TB25 50FRT W 50 Sacace NHS Meningitis Real TM VER 21 03 2013 Sacace NHS Meningitis Real TM VER 21 03 2013 NAME NHS Meningitis Real TM INTENDED USE Kit NHS Meningitis Real TM is a Real Time test for the detection and differentiation of Neisseria meningitidis Haemophilus influenzae and Streptococcus pneumoniae in the biological materials DNA is extracted from specimens amplified using RT amplification and detected using fluorescent reporter dye probes specific for N meningitidis H influenzae S pneumoniae DNA and IC Internal Control PRINCIPLE OF ASSAY NHS Meningitis Real TM Test is based on three major processes isolation of DNA from specimens Real Time amplification of the DNA and NHS Meningitis detection by the polymerase chain reaction PCR based on the amplification of pathogen genome specific region using specific primers and detection via fluorescent dyes These dyes are linked with probes of oligonucleotides which bind specifically to the amplified product The real time PCR monitoring of fluorescence intensities allows the accumulating product detection without reopening of reaction tubes after the PCR run NHS Meningitis Real TM PCR kit is a qualitative test wh
9. cons away from all other reagents and add it to the reaction mix in a separate area e Thaw all components thoroughly at room temperature before starting an assay e When thawed mix the components and centrifuge briefly e Use disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterwards e Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas Do not use a kit after its expiration date e Dispose of all specimens and unused reagents in accordance with local authorities regulations e Specimens should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices e Clean and disinfect all sample or reagent spills using a disinfectant such as 0 5 sodium hypochlorite or other suitable disinfectant Avoid sample or reagent contact with the skin eyes and mucous membranes If skin eyes or mucous membranes come into contact rinse immediately with water and seek medical advice immediately Material Safety Data Sheets MSDS are available on request Use of this product should be limited to personnel trained in the techniques of DNA amplification e The laboratory process must be one directional it should begin in the Extraction Area and then move to the Amplification and Detection Areas Do not return samples equipment and reagents to the area in which the previous
10. ich contain the Internal Control IC It must be used in the isolation procedure in order to control the process of each individual sample extraction and serves also to identify possible reaction inhibition Sacace NHS Meningitis Real TM VER 21 03 2013 MATERIALS PROVIDED Module No 1 Real Time PCR kit B25 50FRT Part N 2 Controls e Negative Control C 1 2 ml e Internal Control IC 1 0 ml e Pos DNAN meningitidis C 0 1 ml e Pos DNA H influenzae C 0 1 ml e Pos DNA S pneumoniae C 0 1 ml e Pos IC C 0 1 ml e DNA buffer 0 5 ml Part N 3 NHS Meningitis Real TM RealTime amplification e PCR mix 1 N meningitidis IC 0 6 ml e PCR mix 1 S pneumoniae H influenzae 0 6 ml e PCR mix 2 2 x 0 3 ml e TaqF Polymerase 2 x 0 03 ml Contains reagents for 55 reactions must be used in the isolation procedure as Negative Control of Extraction 2d add 10 ul of Internal Control during the DNA purification procedure directly to the sample lysis mixture Sacace NHS Meningitis Real TM VER 21 03 2013 Module No 2 Complete Real Time PCR test with DNA purification kit TB25 50FRT Part N 1 DNA Sorb B Sample preparation e Lysis Solution 15 ml e Washing Solution 1 15 ml e Washing Solution 2 50 ml e Sorbent 1 25 ml e DNA eluent 5 0 ml Contains reagents for 50 extractions Part N 2 Controls e Negative Control C 1 2 ml e Internal Control IC 1
11. level should be raised Set the threshold at a level where fluorescence curves are linear and do not cross curves of the negative samples RESULTS ANALYSIS The results are interpreted by the device software through the presence of crossing of fluorescence curve with the threshold line Put the threshold line at such level where curves of fluorescence are linear e Streptococcus pneumoniae is detected on the FAM Green channel and Haemophilus influenzae on the JOE Yellow HEX Cy3 channel with PCR mix 1 S pneumoniae H influenzae e Internal Control IC is detected on the FAM Green channel and Neisseria meningitidis on the JOE Yellow HEX Cy3 channel with PCR mix 1 N meningitidis IC The sample is considered to be positive if the value of Ct is different from zero Ct 40 The sample is considered to be negative if the result is positive only on the channel Fam with PCR mix 1 N meningitidis IC and the Ct value is lower than 40 Ct boundary values Sample Channel Ct CS FAM Green 38 C meningitidis JOE Yellow 38 C s pneumoniae FAM Green 38 C i infiuenzae JOE Yellow 38 C FAM Green 38 Clinical samples FAM Green 38 JOE Yellow 38 Sacace NHS Meningitis Real TM VER 21 03 2013 SPECIFICATIONS Sensitivity DNA Analytical Clinical material extraction PCR kit Pathogen sensitivity kit GE ml Neisseria meningitidis DNA RNA NHS Meningitis Haemophilus
12. lts were not detected Sacace NHS Meningitis Real TM VER 21 03 2013 TROUBLESHOOTING 1 Weak or absent signal of the IC Fam Green channel with PCR mix 1 N meningitidis IC retesting of the sample is required e The PCR was inhibited Make sure that you use a recommended DNA extraction method and follow the manufacturer s instructions Re centrifuge all the tubes before pipetting the extracted DNA for 2 min at maximum speed 12000 16000 g and take carefully supernatant Dont disturb the pellet sorbent inhibit reaction e The reagents storage conditions didn t comply with the instructions Check the storage conditions e The PCR conditions didn t comply with the instructions Check the PCR conditions and for the IC detection select the fluorescence channel reported in the protocol e The IC was not added to the sample during the pipetting of reagents Make attention during the DNA extraction procedure 2 Weak Ct gt 40 sample signal on the Fam Joe Yellow Cy3 HEX channel retesting of the sample is required 3 Joe Yellow Cy3 HEX signal with Negative Control of extraction e Contamination during DNA extraction procedure All samples results are invalid Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol Use only filter tips during the extraction procedure Change tips among tubes Repeat the RNA extraction with the new set of reagents 4 Any signal with Negative PCR Control
13. step was performed Some components of this kit contain sodium azide as a preservative Do not use N metal tubing for reagent transfer Only for Module No 2 Sacace NHS Meningitis Real TM VER 21 03 2013 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 SAMPLE COLLECTION STORAGE AND TRANSPORT NHS Meningitis Real TM can analyze DNA extracted with DNA Sorb B Sacace REF K 1 1 B from e liquor ready for extraction 0 1 ml Specimens can be stored at 2 8 C for no longer than 12 hours or frozen at 20 C to 80 C Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents DNA ISOLATION The following kits are recommended c DNA Sorb B Sacace REF K 1 1 B c DNA RNA Prep Sacace REF K 2 9 Please carry out the DNA extraction according to the manufacturer s instructions Add 10 ul of Internal Control during the DNA isolation procedure directly to the sample lysis mixture Sacace NHS Meningitis Real TM VER 21 03 2013 SPECIMEN AND REAGENT PREPARAT
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