Salmonella spp Real TM PCR ver 21032013 - bio
Contents
1. Vortex vigorously Sorbent and add 25 pl 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 pl 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 13 16 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 Salmonella spp Real TM VER 21 03 2013 PROTOCOL Reaction volume 25 ul Total reaction volume is 25 ul the volume of DNA sample is 10 pl 1 Pre2. 96 Sacace iQ5 iQ iCycler BioRad USA Mx3000P Mx3005P Stratagene USA Applied Biosystems 7300 7500 Real Time PCR Applera SmartCycler Cepheid LineGene K Bioer Eco Real Time PCR System Illumina Sacace Salmonella spp Real TM VER 21 03 2013 RESULTS ANALYSIS 1 The sample is considered to be positive for Salmonella spp 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 Salmonella spp is detected on the JOE Yellow HEX Cy3 channel if in the channel Joe Yellow HEX Cy3 the value of Ct is different from zero Ct lt 40 The sample is considered to be negative if in the channel Joe Yellow HEX Cy3 for Salmonella spp 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 40 Occurrence of any value Ct in the table of results 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 Cont
3. follow to the manufacturer s instructions 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 gt 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 an
4. microcentrifuge for eppendorf type tubes RCF max 16 000 x g 60 C 5 C dry heat block Vortex mixer Pipettors capacity 5 40 ul 40 200 pl 200 1000 pl with aerosol barrier 1 5 ml polypropylene sterile tubes Disposable gloves powderless Biohazard waste container Zone 2 RT and amplification Real Time Thermalcycler Workstation Pipettes Tips with filter Tube racks STORAGE INSTRUCTIONS DNA Sorb B must be stored at 2 8 C Salmonella spp Real TM must be stored at 20 C The complete kit can be shipped at 2 8 C but should be stored at 20 C immediately on receipt STABILITY Salmonella spp 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 Salmonella spp Real TM WARNINGS AND PRECAUTIONS The user should always pay attention to the following o A Lysis Solution contains guanidine thiocyanate Guanidine thiocyanate is harmful if inhaled or comes into contact with skin or if swallowe
5. sop Real TM VER 21 03 2013 MATERIALS PROVIDED Module No 1 Real Time PCR kit B11 50FRT Part N 2 Salmonella spp Real TM Real Time amplification kit e PCR mix 1 Salmonella spp 0 6 ml e PCR mix 2 Flu 0 3 ml e TaqF Polymerase 0 03 ml e Positive Control Salmonella 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 TB11 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 Salmonella spp Real TM Real Time amplification kit e PCR mix 1 Salmonella spp 0 6 ml e PCR mix 2 Flu 0 3 ml e TaqF Polymerase 0 03 ml e Positive Control Salmonella 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 Salmonella spp Real TM VER 21 03 2013 MATERIALS REQUIRED BUT NOT PROVIDED Zone 1 sample preparation DNA extraction kit module No 1 Biological cabinet Desktop
6. _iSacace BIOTECHNOLOGIES REF REF Salmonella spp Real TM Handbook Real Time PCR Kit for detection of Salmonella species B11 50FRT TB11 50FRT Y 50 Sacacet M Salmonella spp Real TM VER 21 03 2013 NAME Salmonella spp Real TM INTRODUCTION Acute Intestinal Infection A l 1 are one of the primary causes of hospitalization in infectious disease departments In accordance with the data provided by the contemporary literature the most often detectable and generally spread etiological agents of A l l are bacterial microorganisms such as Shigella spp and enteroinvasive E coli EIEC Salmonella spp thermophilic group of Campylobacter spp enteropathogenic E coli EPEC and enteroaggregative E coli EAEC and viral agents such as group A rotaviruses genotype 2 noroviruses group F adenoviruses type 40 and 41 and astroviruses INTENDED USE The Salmonella spp Real TM is a Real Time PCR test for the qualitative detection of Salmonella spp in the liquid cultures water and feces PRINCIPLE OF ASSAY Kit Salmonella spp 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 Salmonella spp 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 Salmonella
7. cation 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 Salmonella sop 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 Salmonella spp Real TM can analyze DNA extracted with 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 betwe
8. d 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 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 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 amplifi
9. d instruments with sodium hypochlorite and ethanol or special DNA decontamination reagents Pipette the Positive control at last Repeat the PCR preparation with the new set of reagents Sacace Salmonella spp Real TM VER 21 03 2013 KEY TO SYMBOLS USED List Number Caution Contains sufficient LOT Lot Number for lt n gt tests po Expiration Date VER Version Store at NCA Regalo ONONO 41 Amplification wal Manufacturer NCE Din canun or xtraction li Consult instructions Positive Control of C asce for use Amplification IC Internal Control RUO For Research Use Only SaCycler is a registered trademark of Sacace Biotechnologies Cycler 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 LightCycler is a registered trademark of Roche LineGene K is a registered trademark of Bioer Eco PCR Real Time System is a registered trademark of Illumina Sacace Biotechnologies Srl via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com CERMET is Sacace Salmonella spp Real TM VER 21 03 2013
10. en 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 gt DNA RNA Prep Sacace REF K 2 9 Please carry out RNA extraction according to the manufacture s instruction Add 10 ul of Internal Control during DNA isolation procedure directly to the sample lysis mixture Sacace Salmonella spp Real TM VER 21 03 2013 SPECIMEN AND REAGENT PREPARATION 1 ar wo Nn D 10 11 12 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 pl of Lysis Solution and 10 pl of IC Add 100 pl of Samples to the appropriate tube Prepare Controls as follows e add 100 pl of C Negative Control to labeled Cneg Vortex the tubes incubate 5 min at 65 C and centrifuge for 5 sec
11. ic 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 Nonspecific reactions were absent while testing human DNA samples and DNA panel of the following microorganisms 18 strains of different serogroups of Salmonella spp 3 strains of Cronobacter sakazakii 4 strains of Enterobacter cloacae 2 strains of Enterobacter aerogenes 2 strains of Pantoea agglomerans 8 strains of Campylobacter spp C jejuni C coli and C fetus fetus 31 strains of different serogroups of Esherichia coli including EHEC EPEC ETEC EAggEC and EIEC 12 strains of different species and serogroups of Shigella spp 22 strains of different species and serogroups of Yersinia spp Citrobacter freundii Clostridium perfringens Klebsiella pneumonia Listeria monocytogenes Protrus mirabilis Pseudomonas aeruginosa Serratia marcessens Analytical sensitivity The kit Salmonella spp Real TM allows to detect Salmonella Spp DNA in 100 of the tests with a sensitivity of not less than 1000 copies ml The detection was carried out on the control standard and its dilutions by negative sample Sacace Salmonella spp Real TM VER 21 03 2013 TROUBLESHOOTING 1 Weakorno signal of the IC FAM green channel for the Negative Control of extraction e The PCR was inhibited gt Make sure that you use a recommended DNA extraction method and
12. pare required quantity of reaction tubes for samples and controls 2 Prepare the reaction mix for required number of samples 3 For N reactions mix in a new tube 10 N 1 pl of RT PCR mix 1 Salmonella spp 5 0 N 1 ul of PCR mix 2 0 5 N 1 ul 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 ul of Salmonella IC C to the tube labeled C saimonenaic Amplification 1 Create a temperature profile on your instrument as follows Rotor type instruments Plate type or modular instruments Fluorescence Cycle 3 Fluorescence Cycle SECO vema mime detection repeats TED E Mine detection repeats Hold 95 15 min 1 95 15 min 1 95 10s 95 10s Cycling FAM Green 30 s FAM 2 90 25S JOE Yellow 45 eo JOE HEX Cy3 45 72 10s 72 10s For example Rotor Gene 3000 6000 Corbett Research Australia For example SaCycler
13. rol 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 40 a retesting of the sample is required Table 1 Results for controls Ct channel Fam Ct channel Joe Conirol Stage for control Green Yellow HEX Cy3 Interpretation NCE DNA isolation Pos lt 40 Neg Valid result NCA Amplification Neg Neg Valid result Salmonella sonnei IC C Amplification Pos lt 40 Pos lt 40 Valid result Sacace Salmonella spp 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 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 PERFORMANCE CHARACTERISTICS Analytical specificity The analytical specificity of Salmonella spp Real TM PCR kit is ensured by selection of specif
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