EHEC Real TM ver 21032013
Contents
1. and ethanol or special DNA decontamination reagents Pipette the Positive control at last Repeat the PCR preparation with the new set of reagents Sacace EHEC Real TM VER 21 03 2013 KEY TO SYMBOLS USED List Number Caution Contains sufficient LOT Lot Number for lt n gt tests E Expiration Date VER Version Negative Control of J Store at NCA ganes Amplification Negative control of Manufacturer NCE gaily Extraction Cj Consult instructions C Positive Control of for use Amplification IC Internal Control RUO For Research Use Only SaCycler is a registered trademark of Sacace Biotechnologies iCycler and iQ5 are trademarks of Bio Rad Laboratories Rotor Gene Technology is a registered trademark of Corbett Research MX3000P and MX3005P are trademarks of Stratagene Applied Biosystems is trademarks of Applera Corporation SmartCycler is a registered trademark of Cepheid A Sacace Biotechnologies Srl E N via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com a 9001 2008 Sacace EHEC Real TM VER 21 03 20132. PREPARATION 1 ar wo Nn o NoD 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 ul of Samples to the appropriate tube Prepare Controls as follows e add 100 pul 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
3. e Positive Control EHEC IC 0 1 ml e Negative Control C 1 2 ml e Internal Control IC 1 0 ml e DNA buffer 0 5 ml Contains reagents for 55 tests must be used in the isolation procedure as Negative Control of Extraction add 10 ul of Internal Control during the DNA isolation directly to the sample lysis mixture see DNA Sorb B REA K 1 1 B protocol Sacace EHEC 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 e 60 C 5 dry heat block e Vortex mixer e Pipettors capacity 5 40 ul 40 200 ul 200 1000 ul with aerosol barrier e 1 5 ml polypropylene sterile tubes e Disposable gloves powderless e Biohazard waste container Zone 2 RT and amplification e Real Time Thermalcycler e Workstation e Pipettes e Tips with filter e Tube racks STORAGE INSTRUCTIONS Part N 1 DNA Sorb B must be stored at 2 8 C Part N 2 EHEC Real TM must be stored at 20 C The complete kit can be shipped at 2 8 C but should be stored at 2 8 C and 20 C immediately on receipt STABILITY EHEC 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
4. 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 EHEC Real TM VER 21 03 2013 WARNINGS AND PRECAUTIONS The user should always pay attention to the following gt A 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 amplicons 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 e 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 pote
5. Incubate all tubes with open cap for 5 min at 65 C Resuspend the pellet in 50 pl 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 Only for Module No 2 Sacace EHEC Real TM VER 21 03 2013 PROTOCOL Reaction volume 25 ul Total reaction volume is 25 ul the volume of DNA sample is 10 ul 1 2 3 O Prepare required quantity of reaction tubes for samples and controls Prepare the reaction mix for required number of samples For N reactions mix in a new tube 10 N 1 ul of RT PCR mix 1 EHEC 5 0 N 1 pl of PCR mix 2 0 5 N 1 ul of TaqF Polymerase Vortex the tube then centrifuge shortly Add 15 wl of prepared reaction mix into each appropriate tube Using tips with aerosol filter add 10 wl 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 Prepare for each panel 3 controls Amplification add 10 pl of DNA buffer to the tube labeled Amplification Negative Control add 10 pl of EHEC IC C to the tube labeled C eyecyc 1 Create a t
6. _iSacace BIOTECHNOLOGIES REF REF EHEC Real TM Handbook Real Time PCR Kit for detection of EHEC Enterohemorrhagic E coll B59 50FRT TB59 50FRT 50 M EHEC Real TM VER 21 03 2013 NAME EHEC Real TM INTRODUCTION EHEC Real TM kit is for the qualitative detection of Shiga toxins 1 and 2 also called Verotoxins produced by E coli in cultures derived from clinical stool specimens EHEC Real TM kit is used in conjunction with the patient s clinical symptoms and other laboratory tests to aid in the diagnosis of diseases caused by enterohemorrhagic E coli EHEC infections Shiga toxins can be classified into two main categories Shiga toxin 1 ST1 and Shiga toxin 2 ST2 EHEC strains may produce ST1 or ST2 only or both ST1 and ST2 simultaneously EHEC are capable of initiating life threatening illnesses particularly in young children the elderly or patients with immune deficiency The main sources of infection are contaminated raw or insufficiently heated foods of animal origin e g meat and dairy products The reservoirs for EHEC are cattle sheep and goats and it is spread through their feces These microorganisms can enter food during the processing of meat and dairy products if hygienic conditions are inadequate The incidence of food infection caused by Shiga toxin producing E coli demands reliable and rapid methods of detection In addition to traditional culture methods molecular biology tec
7. ation the amplification and the detection steps are performed correctly lf 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 PERFORMANCE CHARACTERISTICS Analytical specificity The analytical specificity of the primers and probes was validated with negative samples They did not generate any signal with the specific EHEC primers and probes The specificity of the kit was 100 The potential cross reactivity of the kit was tested against the group control Esherichia coli EPEC ETEC EAggEC EIEC Cronobacter sakazakii Enterobacter cloacae Enterobacter aerogenes Pantoea agglomerans Campylobacter spp C jejuni C coli C fetus fetus Salmomella spp Yersinia spp Citrobacter freundii Clostridium perfringens Klebsiella pneumoniae Listeria monocytogenes Protrus mirabilis Pseudomonas aeruginosa Serratia marcessens It was not observed any cross reactivity with other pathogens Analytical sensitivity The kit EHEC Real TM allows to detect EHEC DNA in 100 of the tests with a sensitivity of not less than 1000 copies ml Sacace EHEC Real TM VER 21 03 2013 TROUBLESHOOTING 1 Weak or no signal of the IC FAM green channel for the Negative Control of extraction e The PCR was inhibited Make sure that you use a recommended DNA extraction method and follow to the manufacturer s instr
8. emperature profile on your instrument as follows Rotor type instruments Plate type or modular instruments 3 Fluorescence Cycle 5 Fluorescence Cycle Sece rema die detection repeats TEIND E Mine detection repeats Hold 95 15 min 1 95 15 min a 1 95 10s 95 10s Cycling FAM Green 30s FAM 2 i 25s JOE Yelow 5 6p JOE HEX Cy3 5 72 10s 72 10s 7 For example Rotor Gene 3000 6000 Corbett Research Australia 2 For example SaCycler 96 Sacace iQ5 iQ iCycler BioRad USA Mx3000P Mx3005P Stratagene USA Applied Biosystems 7300 7500 Real Time PCR Applera SmartCycler Cepheid Sacace EHEC Real TM VER 21 03 2013 RESULTS ANALYSIS 1 The results are interpreted by the device software through the presence of crossing of fluorescence curve with the threshold line e IC DNA is detected on the FAM Green channel e EHEC is detected on the JOE Yellow HEX Cy3 channel The sample is considered to be positive for EHEC if in the channel Joe Yellow HEX Cy3 the value of Ct is different from zero Ct lt 35 The sample is considered to be negative if in the channel Joe Yellow HEX Cy3 for EHEC if the Ct value is not determined the fluorescence curve does not cross the threshold line and in the results table on the channel Fam Green value for Internal Control is lower than 33 Occurrence of any value Ct in the table of resul
9. h DNA Sorb B from e Liquid cultures e water centrifuge 10 20 ml for 10 min at maximum speed Discard the supernatant and leave about 100 ul of solution for DNA extraction e whole blood collected in EDTA tubes e feces gt Prepare 20 feces suspension by adding in 5 ml tube of 4ml of Saline Solution and 1 0 gr approx 1 0 ml of feces Vortex to get the homogeneous suspension and centrifuge for 5 min to 7000 12000g and using a micropipette with a plugged aerosol barrier tip transfer in a new sterile 1 5 ml tube 100 ul of the bacterial fraction white yellowish line between the sediment and the supernatant gt Add 800 ul of PBS or Saline Solution Vortex to get the homogeneous suspension and centrifuge for 5 min to 7000 12000g Remove and discard the supernatant gt Resuspend the pellet in 0 3 ml of PBS or Saline Solution It is recommended to process samples immediately after collection Store samples at 2 8 C for no longer than 24 hours or freeze at 20 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 kit is recommended gt DNA Sorb B Sacace REF K 1 1 B Please carry out DNA extraction according to the manufacture s instruction Add 10 ul of Internal Control during DNA isolation procedure directly to the sample lysis mixture Sacace EHEC Real TM VER 21 03 2013 SPECIMEN AND REAGENT
10. hniques are becoming more useful due to their improved specificity and sensitivity INTENDED USE The EHEC Real TM is a Real Time PCR test for the qualitative detection of EHEC in the liquid cultures water and feces PRINCIPLE OF ASSAY Kit EHEC Real TM is based on two major processes isolation of DNA extracted from samples and amplification by real time PCR with fluorescent reporter dye probes specific for EHEC and Internal Control IC Test contains an IC which serves as an amplification control for each individually processed specimen and to identify possible reaction inhibition Sacace EHEC Real TM VER 21 03 2013 MATERIALS PROVIDED Module No 1 Real Time PCR kit B59 50FRT Part N 2 EHEC Real TM Real Time amplification kit e PCR mix 1 EHEC 0 6 ml e PCR mix 2 Flu 0 3 ml e TaqF Polymerase 0 03 ml e Positive Control EHEC IC 0 1 ml e Negative Control C 1 2 ml e Internal Control IC 1 0 ml e DNA buffer 0 5 ml Contains reagents for 55 tests Module No 2 Complete Real Time PCR test with DNA purification kit TB59 50FRT Part N 1 DNA Sorb B Sample preparation kit 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 EHEC Real TM Real Time amplification kit e PCR mix 1 EHEC 0 6 ml e PCR mix 2 Flu 0 3 ml e TaqF Polymerase 0 03 ml
11. ntially 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 e 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 e Material Safety Data Sheets MSDS are available on request e 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 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 EHEC Real TM VER 21 03 2013 PRODUCT USE LIMITATIONS 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 EHEC Real TM can analyze DNA extracted wit
12. ts for the negative control sample on the Joe Yellow HEX Cy3 channel and for negative control of amplification DNA buffer on any of channels testifies contamination of reagents or samples In this case results of the analysis for all tests are considered invalid It is required to repeat the analysis of all tests and also to take measures to detect and eliminate the source of contamination No signal with Positive Control indicates incorrect programming of the Real Time instrument repeat the amplification with correct setting If the Ct value of the Internal Control is absent or higher than 33 a retesting of the sample is required Table 1 Results for controls Ct channel Fam Ct channel Joe Control Stage for control Green Yellow HEX Cy3 Interpretation NCS DNA isolation Pos lt 33 Neg Valid result DNA buffer Amplification Neg Neg Valid result EHECI IC C Amplification Pos lt 33 Pos lt 33 Valid result Sacace EHEC Real TM VER 21 03 2013 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 negative amplification control NCA positive amplification control C are required for every run to verify that the specimen prepar
13. uctions Re centrifuge all the tubes before pipetting of the extracted DNA for 2 min at maximum speed 12000 16000 g and take carefully supernatant Don t disturb the pellet sorbent inhibit reaction e The reagents storage conditions didn t comply with the instructions Check the storage conditions e Improper DNA extraction Repeat analysis starting from the DNA extraction stage e The PCR conditions didn t comply with the instructions Check the PCR conditions and select for the IC detection 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 or no signal of the Positive Control e The PCR conditions didn t comply with the instructions Check the amplification protocol and select the fluorescence channel reported in the manual 3 JOE Yellow HEX Cy3 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 between tubes Repeat the DNA extraction with the new set of reagents 4 Any signal with Negative Control of PCR DNA buffer e Contamination during PCR preparation procedure All samples results are invalid Decontaminate all surfaces and instruments with sodium hypochlorite
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