User manual Premi®Test Salmonella
Contents
1. Place the colony sampler in the 100 ul Lysis Buffer and twist the sampler between thumb and index finger while remaining in the buffer Remove the sampler and discard Close the tube vortex and continue with the lysis step 2 Is Transfer the 1 5 ml tubes with the resuspended cells to a heating block and incubate at 99 C for 15 min For this purpose the Thermomixer may be used set the Thermomixer to 99 C This heat treatment disrupts the cells and releases the DNA into the Lysis Buffer 6 2 Vortex and cool down to room temperature by placing the tubes on the table When the samples aren t used directly store the tubes at 20 C 3 Thaw and vortex before continuing with step 3 3 DNA recognition step A Procedure Make sure to always use a new pipet tip when adding solutions or samples to a reaction tube to avoid contamination 1 First add 5 ul solution A cap colour amber to every reaction tube of the strip supplied with the kit Next add 10 ul of DNA extract step 2 of each sample Please write down the sample reference for each tube of the strip 2 Close the tubes and spin down briefly using the minifuge to collect both sample and solution A at the bottom of the tubes Mix well by tapping against each strip the solution should have a uniform blue colour Again spin down briefly using the minifuge 3 Place the strip s in the thermocycler and run the CP step A program total sample volume 18 ul The prog2. The Premi Test Salmonella has been developed to serotype Salmonella enterica subsp enterica bacteria The test also detects the other Salmonella species and subspecies but does not discriminate all species Consequently the test indicates the presence of Salmonella in these cases as a genovar score without a specific serotype except for Salmonella bongori Other biochemical tests are required to identify these strains Specific Salmonella serotypes originating from certain feed and food matrices may give suboptimal results with Premi Test Salmonella in case of sampling from MSRV Not all Salmonella serotypes have been extensively validated within all food and feed matrices with an MSRV pretreatment Therefore an internal validation with the most occurring serotypes in the common food or feed matrices is recommended The Premi Test Salmonella is able to fully serotype non motile or monophasic Salmonella So for example a monophasic S Typhimurium strain will be typed as S Typhimurium A specific exception is the S 1 4 5 12 i When the specific DNA marker for the FIjA gene is absent the score will be the S 1 4 5 12 i In other cases S 1 4 5 12 i strains are identified as S Typhimurium For known overlapping scores the probability score of the dominant serotype is based on the prevalence of the respective serotypes according to the database of the Global Salmonella Survey of the World Health Organization WHO http www who
3. Picture not found or Image capture error Check if the AT reader is properly installed Please send an e mail to Technical Support if reinstallation of the reader does not solve this problem The AT image is covered with small spots There may be various causes for this phenomenon Most likely the array dried out during the detection step Please be sure that the AT always contains sufficient amounts of reagents Detection Buffer Blocking Buffer conjugate dilution or Staining Solution This is particularly critical during the 18 incubations steps at 50 C In most cases the software will be able to handle these small spots If not repeat the detection step with a new AT 12 The AT was incubated for more than 15 minutes with Staining Solution before taking the image Results may be unreliable due to overstaining Inspect image if spots are very dark please repeat detection step with a new AT 13 Duplo sample does not display identical result 13 1 Inspect images for dust particles Tap or gently shake the tubes to try to remove the particles from the array and rescan the images 13 2 In case of extra spots repeat the test to confirm result 14 wish to stop the assay and continue at a later stage What is the best point to stop the assay You may stop the reactions after steps B or C Store reaction mixtures in that case at 4 C when used within 24 hours or at 20 C for a maximum period of two weeks 15 The AT i
4. C 2 min Holding at 4 C 37 C 45 min 95 C 10 min Holding at 4 C 95 C 10 min 95 C 5 sec 55 C 30 sec 72 C 30 sec 98 C 2 min Holding at 4 C 21 Premi Test Salmonella APPENDIX 2 Salmonella serotypes identified with Premi Test Salmonella Panama 74 _ Paratyphi A 6 Altona Paratyphi B v Java Infantis Paratyphi C Pomona 9 Bareilly Poona _80 Pullorum Regent 85_ San Diego 86 Schwarzengrund 17 Carrau Stanley 89 Stourbridge 90 Telekebir Choleraesuis Tennessee 92 Thompson 94 Typhimurium 4 60 Mikawasima 61 96 Virchow 98 Weltevreden 99 Worthington 66 Napoli Gloucester 68 Oho 69 Oranienburg This list will be extended with more serotypes on a regular basis More information can be found on the Check Points website http www check points com products check and trace salmonella serotypes html Serotypes not included in this test may yield a genovar score e g Salmonella genovar 428 1 FSIS notice 04 026 February 2006 2 Regulations EC N 1003 2005 and EC N 1168 2006 3 Salmonella serotypes frequently found by EFSA in broiler flocks 4 Most frequently reported Salmonella serotypes from human sources reported to the CDC in 2005 22 APPENDIX 3 Pipetting scheme for B or C mix Pipetting scheme for conjugate dilution ul ul B1 ul B2 conjugate ul or C1 or C2 solution
5. at this stage as the database cannot be edited once the results have been read Check Points 1 Array selection 2 DNA Recognition step A B and C 3 Detection step 4 Results 2 DNA Recognition step A B and C iS Premi i st Salmonella Please fill in your sample codes in the table below Sample information Customer Enrichment Extraction Source Remarks 12 13 14 15 16 17 18 19 20 21 22 23 24 E Fi H H E E E EB E RE lt E H Ent n E Figure 5 Pop up for additional sample details 13 SPremi Test Salmonella f Proceed to the Detection step sheet see Figure 6 by clicking the Next Step button g The software will now indicate the samples that need to be analyzed first see figure 6 1 Anay selection 2 DNA Recognition step A B and C 3 Detection step Ea k Points Premi Reader found BM000000 Test Salmonella Please enter ArrayTube LOT number place tube in reader and Confirm Sample s Ref 1 Ref 2 AnayTube LOT number Ret 3 Figure 6 Detection step in the software 17 Enter the AT lot number in the appropriate field when the 15 minutes incubation time is completed Next insert the AT with open cap into the reader close the lid of the reader and click on the Confirm button in the software The Staining Solution remains in the AT while reading The results will be displayed immediately After clicking Save Results the resu
6. where the detection step is carried out Do not open de tubes at the same location as where steps A B and C are performed e Open and close tubes with amplified material very carefully e Use separate equipment pipettes sample holders lab coats gloves disposables and reagents for the detection step then those that are used for the previous steps A B and C Never transfer items from the detection step location to the location where steps A B and C are carried out e Wear a clean lab coat and clean gloves during all steps of the test e Keep the tubes of all kit components and samples closed as much as possible e Clean the lab benches and all equipment regularly with a 0 5 hypochlorite solution SPremi st Salmonella PROTOCOL PREMI TEST SALMONELLA It is strongly recommended that the full protocol is read before using the test The protocol consists of the following steps Sd PS Sampling Lysis DNA recognition step A DNA recognition step B DNA recognition step C Detection step Pure presumptive Salmonella spp culture Non selective agar e qg nutrient agar 1 2 3 Dispense 100 ul Lysis Buffer into a 1 5 ml tube Use a separate tube for each sample Take a colony sampler and pierce through a single colony into the agar Briefly touch the bottom of the plate and take the sampler out again Always keep the colony sampler in a vertical position as shown in the figure below It is advised to wear gloves
7. image is still too dark please repeat the detection step with a new AT The software indicates Reference spots not found The software did not find reference spots on the AT Causes may be 6 1 An air bubble is interfering with the result Tap the AT gently or pipet the liquid gently up and down and then retry to take the image 6 2 The picture is very dark conjugate dilution not removed properly Please refer to question 5 6 3 The picture is completely white Staining Solution was not added Please add Staining Solution again and proceed from detection step 15 6 4 Some spots are missing due to scratching of the array with a pipet tip during adding or removing of solution from or to the AT Please repeat the detection step with a new AT If the results do not improve staining has failed Most likely no conjugate dilution was added or the conjugate dilution was not prepared properly Please repeat detection step with new AT 10 11 SPremi st Salmonella The software indicates Hybridisation not OK The software did not find the hybridisation control spots on the AT The hybridisation control is used to check if the hybridisation at 50 C of the PCR product with the AT has been performed properly Causes may be 7 1 The picture is completely dark conjugate dilution not removed properly Please refer to question 5 7 2 The reaction mixture after completion of step C was not added to the AT Please r
8. int salmsurv en In some cases Premi Test Salmonella may yield two serovar names due to a known overlap in genovar score or due to historically given names to serovars containing the same global antigenic formula As an example of the latter S Paratyphi C and S Choleraesuis are considered to be part of the same group 6 7 c 1 5 This group also contains Choleraesuis var Kunzendorf var Decatur and Typhisuis which serotypes have not yet been validated in the Premi Test Salmonella Therefore the result for this group will now read S Paratyphi C or S Choleraesuis More details about the overlap in genovar scores as well as frequent updates of the Serotype list appendix 2 can be found on the Check Points website http www check points com products check and trace salmonella serotypes html 1 Wattiau P et al Evaluation of the Premi Test Salmonella a commercial low density DNA microarray system intended for routine identification and typing of Salmonella enterica International Journal of Food Microbiology 2008 Vol 123 p 293 298 2 Wattiau P et al Comparison of Classical Serotyping and PremiTest Assay for Routine Identification of Common Salmonella enterica Serovars J Clin Microbiol 2008 Vol 46 p 403 7 4040 20 APPENDIX 1 Step A Cycle 1 1x 95 C 3 min Cycle 2 24x Cycle 3 1x Step B Cycle 1 1x Step C Cycle 1 1x Cycle 2 35x Cycle 3 1x 95 C 30 sec 65 C 5 min 98
9. step A is finished 3 Briefly spin down the reaction tubes after step A is finished 4 Add 15 ul of the freshly prepared B mix to each sample in the strip s Close the tubes mix by tapping each strip and spin down briefly 5 Place the strip s in the thermocycler and run the CP step B program total sample volume 33 ul The program will run for approximately 1hour The step B program is outlined in appendix 1 Note B2 is a glycerol based solution and is therefore not frozen and ready for use immediately 5 DNA recognition step C Procedure 14 Approximately 10 minutes before the end of step B Take solution C1 cap colour yellow from the freezer Thaw properly at room temperatuur mix well and spin down briefly Then take solution C2 cap colour red from the freezer and prepare a C mix in a 1 5 ml tube using the pippetting scheme appendix 3 at the back of the protocol First add the required amount of C1 solution to the tube Then dispense solution C2 in solution C1 by pipetting up and down 3 times Mix very well by vortexing and spin down briefly 2 Briefly spin down the reaction tubes after step B is finished 3 Add 15 ul of the freshly prepared C mix to each sample in the strip s Close the tubes mix by tapping each strip and spin down briefly 4 Place the strip s in the thermocycler and run the CP step C program total sample volume 48 ul The program will run for approximately 1 5 hour The s
10. 15 The conjugate solution is stored at 20 C but is not frozen and can be used immediately Conjugate dilutions have to be made fresh and should be used on the day of preparation Remove the Blocking Buffer completely and add 150 ul of the conjugate dilution Incubate for 15 minutes at 30 C 400 rpm Remove the conjugate dilution from the ATs and add 600 ul of Detection Buffer Shake the tubes for 2 minutes at 30 C 400 rpm Replace the Detection Buffer by 600 ul of fresh Detection Buffer and shake the tubes again for 2 minutes at 30 C 400 rpm Remove the Detection Buffer from the ATs and add 150 ul of Staining Solution to each AT Incubate for 15 minutes at room temperature to complete the staining procedure Continue with the image analysis immediately after the 15 minutes incubation time Do not incubate with Staining Solution for more then 15 minutes images may get too dark During the 15 minutes incubation complete the required sample information and relevant test data in the Check Points software as outlined below Note Store the bottle with Staining Solution in the dark after use SPremi st Salmonella 16 Filling out the experimental data a Start computer b Start software on the computer by double clicking the desktop icon PTS c Double click on PremiTest Salmonella arr in sheet 1 Array selection of the software as shown in Figure 2 z3 Check Points 1 Anay selection 2 DNA Recognitio
11. AND HANDLING The components of the kit must be stored at 20 C and room temperature respectively Please check the individual components for optimal storage conditions immediately after delivery of the kit Reagents stored at the appropriate storage conditions can be used until the expiration date indicated on the boxes Please inspect visually the unopened boxes to ensure that their content is intact Do not use the products when damaged Please contact the Check Points office at serovar check points com if you have any questions and in case shipping has taken more than 3 days SPremi st Salmonella MATERIALS REQUIRED BUT NOT SUPPLIED WITH THE KIT Two different starter sets are available for Premi Test Salmonella Basic starter set Micro Array Reader ATR 03 Check Points software Total starter set Basic starter set PCR instrument thermocycler Thermomixer Eppendorf Comfort with thermoblock for 1 5ml tubes Equipment not supplied Vortex mixer Personal computer Two bench top microfuges for PCR tubes spectrafuge mini Supplies Disposable laboratory powder free gloves Pipettes amp disposable tips preferably filtertips for volumes of 1 to 1000 ul 1 5 ml tubes Eppendorf 10 ml tubes Non selective isolation agar nutrient agar PRECAUTIONS AND RECOMMENDATIONS FOR BEST RESULTS e The test must be performed by adequately trained personnel e Food samples and enrichment culture
12. Det Buf Fg AB oon B OR I 1 1 495 990 990 1485 1980 34 36 540 3 1980 630 E 2475 720 2970 810 2970 1158 60 900 n j 3960 Despite the utmost care in the development and preparation of the protocol Check Points cannot take any responsibility for any errors omissions and or future changes herein 23
13. Premi Test Salmonella version 8 1 Issued February 21 2011 www check points com User manual PremieTest Salmonella Routine Salmonella Serotype Identification Cat No 10 0010 PERFORMANCE TESTED V RESEARCH INSTITUTE i WWW CHECK POINTS COM LICENCE NUMBER 121001 Check amp Trace Salmonella TABLE OF CONTENTS Intended use Principle of the method Kit components Shelf life Storage and handling Materials required but not supplied Precautions and recommendations for best results Protocol Sampling Lysis DNA recognition step A DNA recognition step B DNA recognition step C Detection step FAQ Limitations Appendix 1 Appendix 2 Appendix 3 Developed and produced by Check Points BV Binnenhaven 5 6709 PD Wageningen THE NETHERLANDS Website www check points com E mail info check points com OONNOAOOK KR WWDHDN ND SPremi st Salmonella INTENDED USE The Premi Test Salmonella 10 0010 is designed for the rapid molecular confirmation and serotyping of presumptive Salmonella spp The test employs highly specific DNA markers to allow accurate identification of the Salmonella serotype when Salmonella spp is present The Salmonella serotypes currently identified are shown in appendix 2 PRINCIPLE OF THE METHOD The principle of the Premi Test Salmonella system is based on specific molecular recognition of DNA target sequences and subsequent amplification with universal primers Each single DNA targe
14. Premi st Salmonella 4 Remove the Detection Buffer from the ATs and repeat step 3 5 Replace the Detection Buffer by 300 ul of fresh Detection Buffer 6 Take the samples from step C Samples stored longer than 2 hours after step C was finished should be heated in the Thermocycler at 95 C for 2 minutes Briefly spin down the reaction mixture 7 Transfer 10 ul reaction mixture from each tube of one strip to the corresponding AT in total 30 ul per AT The total volume of the AT will be 330 Ul The lid of the AT should be labelled for reference Caution Samples may contain a white coloured precipitate This is due to denaturation of one of the reaction components a protein stabilizer The presence of this precipitate has no effect on the result of the detection step and may be ignored when adding the sample When adding samples to the AT do not remove the AT from the Thermomixer to prevent the buffer from cooling down Add the sample directly into the Detection Buffer of the AT by pipetting up and down With the completion of step 7 three samples have been added to one AT 8 Close the lids of the ATs and shake the tubes for 30 minutes at 50 C 400 rpm Caution Close the lid of the AT properly to prevent the AT from drying out 9 After 30 minutes remove the complete AT content Detection Buffer with samples and replace by 300 Ul of Blocking Buffer do this with one AT at a time Remove the AT from the Thermomixe
15. epeat detection step with a new AT 7 3 Hybridisation temperature too high Please verify that the thermomixer temperature has been at 50 C when the ATs were hybridised Please repeat detection step with a new AT 7 4 The C mix was not prepared properly or was not added to the assay please repeat the test from step A The software indicates DNA recognition not OK The software did not find the reaction control spots on the AT These reaction controls are used to check the performance of the assay in steps A and C Possible explanations are 8 1 The sample DNA was not added to the assay in step A Please repeat the test 8 2 The sample DNA contains contaminants inhibiting the reactions Please redo the Lysis and repeat the test 8 3 The A mix was not added in step A Please repeat the test 8 4 The C mix was not prepared properly or was not added to the assay please repeat the test 8 5 Reaction mixtures from step C step 7 of detection step were not all added to the AT 3 reaction mixtures should be added one or two may be omitted The software indicates Salmonella suspected The software did not found sufficient spots to give a conclusive result Please inspect the picture visually an air bubble or dust particles may interfere with the result Tap the AT gently or pipet the liquid gently up and down and retry to take the image Please repeat the test if the results do not change The software indicates
16. lts will be saved in the database The software will now indicate which sample should be analysed next Repeat this step until all ATs are analysed 1 Array selection 2 DNA Recognition step A B and C ion s 4 Results Scan ready 3 Detection step FIA IPr emi Reader found BM000000 est Temi lla Please enter ArrayTube LOT number place tube in reader and Confirm Sample s Refi Rel 2 ArtayTube LOT number Ref 3 Confirm C a h r er O i SI of terar a CO o_o Figure 7 Presentation of the final result of an ArrayTube in the software 14 Note 18 Clicking Confirm leads to immediate scanning of the AT Only click Confirm after the full incubation period of 15 minutes is completed If the AT is not in the reader after clicking the Confirm button simply put the AT in the reader and click Confirm again It is important to adhere to the 15 minutes incubations time with Staining Solution as much as possible step 15 A shorter incubation time may lead to faint images exceeding the 15 minutes incubation time may lead to overstaining In both cases incorrect results may be obtained When all ATs are analysed a new window with the summary of the results will appear which can also be printed using the Print button Click the Quit button to end this run of analyses Note For support concerning results please send the result image along with the desired information to ser
17. mage contains dust particles The software will correct this in most cases To prevent any interference with the results please take the AT out of the reader and shake it gently until the dust particles have moved to the side of the AT For technical support please send an e mail to serovar check points com Premi Test Salmonella LIMITATIONS OF THE TEST Premi Test Salmonella uses highly specific DNA markers to identify the Salmonella serotype The presence or absence of a range of these markers determines the serotype The correlation between this DNA marker profile expressed as genovar score and the serotype has been tested and validated extensively for the serotypes listed in appendix 2 1 2 unpublished results Additional work with a wide range of serotypes has indicated that the vast majority of serotypes yield a unique genovar score The Premi Test Salmonella has been tested against many but not all serotypes lt cannot be excluded that certain exotic serotypes generate an overlapping genovar score with another serotype as more than 2600 Salmonella serotypes have been described in literature Therefore the Premi Test Salmonella cannot and does not make any representation or warranty that the Premi Test Salmonella is capable of detecting every species subspecies or serotype of the salmonella genus in any sample source Results may need to be confirmed by traditional methodology in specific cases e g for regulatory samples
18. n step A B and C 3 Detection step 1 Array selection Check Points DPr em est Salmonella Please choose array type from the list below PremiTest Salmonella arr PTS defmatnon 13052009 __ __ Please enter lot number of the kit Operator Figure 2 screen 1 Array Selection d Enter the lot number of the kit in the appropriate field optional and the name of the Operator optional followed by clicking the Next Step button Ref 1 Figure 3 Ref 2 7 Ref 3 Example of filling out sample references in the software and assigning samples to the corresponding tubes Ref 1 Hef 2 Ref 3 e Insert the sample codes in the sheet DNA Recognition A B and C as shown in Figures 3 and 4 Proper analysis is not possible without sample codes Check Points 1 Array selection 2 DNA Recognition step A B and C 3 Detection step 4 Results 2 DNA Recognition step A Band C Check Points SPremi st Salmonella Please fill in your sample codes in the table below Figure 4 screen 2 DNA Recognition A B and C Note Additional remarks may be added per sample optional when sample references have been filled out Double click on one of the sample reference fields A pop up will appear see fig 5 allowing remarks to be added to individual samples or to all samples by checking the box es of the relevant sample s Relevant information of the samples must be added
19. ovar check points com To do this double click on the result s in question in the result summary window A pop up will appear see figure 8 in which your comments may be added to the file li Send Picture DEAR Info Customer Check Points Place Wageningen Country NL Date Time Sample 0504 038 esult Salmonella Schwarzengrund Genovar 1490 Sample esult Salmonella Hadar Genovar 16293 Sample Result Salmonella Telelkebir Genovar 12855 SWVersion PTS2 61 Update List Version Array PTS2 arr Comments Options M Send picture directly over internet connection Save Send later Figure 8 send picture pop up Click on the save send later button to store the file which will have a cpfe extension on the computer and send it by e mail Alternatively if the computer has internet access check the box send picture directly over internet connection see figure 9 and enter the reply e mail address followed by clicking send 15 SPremi st Salmonella In both cases feedback should be expected within three working days Comments Reply email address Options Cancel Send Figure 9 to send pictures directly over the internet check the box Support may also be required in a later stage when the program has been closed For this purpose the send picture pop up may also be accessed from the Database viewer DBview
20. r discard the AT content using a pipette and immediately replace with 300 Ul Blocking Buffer using a new pipette tip Place the AT back in the Thermomixer at 50 C and proceed to the next AT until all the AT solutions have been replaced with Blocking Buffer Shake for 5 minutes at 50 C 400 rpm Caution Transfer the liquids you remove from the ATs to a disposable tube and dispose of it the same day with the other laboratory waste It is important to replace the AT content with blocking buffer one AT at a time Empty ATs in the Thermomixer at 50 C may become very dry thereby increasing the risk of background noise Optional 10 11 Evaporated water condensed on the lid of the AT may be removed with a pipette before removing the Detection Buffer containing the sample Replace the Blocking Buffer with 300 ul of fresh Blocking Buffer Adjust the temperature of the Thermomixer to 30 C and incubate for 10 minutes 400 rpm During this incubation time the Thermomixer can cool down from 50 C to 30 C In the mean time prepare a dilution of the conjugate solution black tube with black cap with Detection Buffer using the pipetting scheme appendix 3 at the back of this protocol For this purpose a 1 5 ml tube or a 10 ml tube may be used depending on the amount required Dispense the conjugate solution in the Detection buffer by pipetting up and down 3 times Mix well by vortexing for 30 seconds Note 12 13 14
21. ram will run for approximately 2 5 hours The step A program is outlined in Appendix 1 Note The reaction tubes supplied with the kit are prefilled with a small amount of blue coloured reagent Proper mixing of this reagent solution A and the sample is crucial for optimal performance Solution A is frozen and should be thawed at room temperature and mixed properly before use When the DNA extracts have been stored at 20 C please thaw the sample properly and mix well When closing the tubes of the strip s don t use excessive pressure as the cap may distort and the sample may then evaporate during the different steps of the protocol Use a new pipette tip for every sample to avoid sample contamination 4 DNA recognition step B Procedure 1 Prepare B mix in a 1 5 ml tube for recognition step B while step A is proceeding Take solution B1 cap colour purple and solution B2 cap colour blue from the freezer Solution B1 is frozen B2 is not and should be thawed properly at room temperature mixed well and spun down briefly before use Use the pipetting scheme appendix 3 at the back of this protocol to prepare the required amount of B mix First add the required amount of B1 solution to the tube Then dispense solution B2 in 7 SPremi st Salmonella solution BI by pipetting up and down 3 times Mix very well by vortexing and spin down briefly 2 Store the B mix in the refrigerator or on ice until
22. s must be treated and discarded as potentially infectious material e Spinning down for a few seconds is done in the various steps to ensure all material is properly collected on the bottom of the tubes e The quality of the results depends on strict compliance with the following good laboratory practice especially concerning PCR Do not use reagents after their expiration date Before use thaw frozen reagents gradually at room temperature and vortex briefly to obtain a homogeneous solution After vortexing briefly spin down the solution to avoid contamination when opening the lid Periodically verify the accuracy and precision of pipettes as well as correct functioning of the instruments Prevention of contamination PCR produces a very high quantity of DNA amplification products amplicons even from minute quantities of starting material The Premi Test Salmonella may therefore yield unreliable results if samples become contaminated with amplicons from previous amplification reactions prior to the PCR step C of the protocol Preventive measures to minimize the risks of amplicon contamination must be taken and are outlined below Caution To prevent contamination we recommend to use pipettes with hydrophobic filter tips Please read carefully and follow these instructions e Keep the detection step separate from recognition step A B and C e After the completion of step C transfer the reaction products to the location
23. shortcut that is located on the desktop Open the database by clicking file followed by open the database is located in C Images by default and scroll down to the results which require support By clicking save the send picture pop up will appear the same as in the result summary window FREQUENTLY ASKED QUESTIONS FAQ 1 The thermocycler states an error in step A B or C Please send an e mail to Technical Support During the different steps step A B or C sample s have partly evaporated Tubes may not have not been closed properly Please restart the procedure from step A I have left Solutions A B and or C out of the 20 C 4 F storage These reagents must be stored at 20 C 4 F for proper performance of the test The performance of the product cannot be fully guaranteed if these solutions were left out of 20 C 4 F for longer then 24 hours Staining Solution turned blue after adding it to the AT Conjugate dilution was not properly removed by washing steps 13 and 14 detection step Continue incubation with Staining Solution for 15 minutes and take the image as described in the protocol If the image is too dark please refer to question 5 The picture of the array is very dark The conjugate dilution detection step 12 was not properly removed by washing steps 13 and 14 detection step Please replace the Staining Solution with Detection buffer and take image immediately If the
24. t is recognized by a specific probe that contains a unique ZIP code corresponding to a unique position address on the microarray These ZIP codes are used for detection on the microarray after amplification Probe ends are joined by a DNA ligase when they match perfectly with the target DNA Only ligated probes will result in amplification products Probes that differ from the target DNA will not give amplification products even in case of a single nucleotide difference Amplification products are hybridized to the microarray and visualized by colorimetric detection The microarrays are contained in so called Array Tubes which are inserted in an Array Tube Reader upon completion of the detection reaction This generates an array image that is analysed by dedicated software to yield a definitive and objective assay result KIT COMPONENTS FOR 72 SAMPLES 9 0020 Colony samplers Room temperature 9 0015 Lysis Buffer Room temperature 9 0007 Detection Buffer Room temperature 9 0008 Blocking Buffer Room temperature 9 0014 Staining Solution 1 bottle 5 ml Ion in the dark 10 0003 Array Tubes ATs Room temperature 9 0010 Manual Not critical 9 0022 Blue tray 24x3 tube strip 2 5 ul reagent tube Positive and negative controls are built into the system It is however strongly recommended to use a positive and negative control for each series of reactions e g a salmonella of a known serotype and a non salmonella strain SHELF LIFE STORAGE
25. tep C program is outlined in appendix 1 Note C2 is a glycerol based solution and is therefore not frozen and immediately ready for use 5 Briefly spin down the reaction mixture after step C is finished and transfer the reaction tubes to the area where the detection step is carried out 6 Store the reaction mixtures at 4 C 1 C when the detection step is carried out within 24 hours Alternatively store the samples at 20 C when the detection step is carried out within two weeks 6 Detection step Figure 1 the Array Tube AT and the Check Points Tube Reader Note The ArrayTube DNA microarray platform is sold under licence from Alere Technologies GmbH Procedure 1 Start preparing the required number of Array Tubes ATs for detection approximately 10 minutes before the end of the step C program Heat the Thermomixer to 50 C Note One AT is required for every 3 tube strip 2 Remove the ATs from their package s and place them in the Thermomixer at 50 C 3 Add 300 ul of Detection Buffer to every AT and shake the tubes for 2 minutes 400 rpm in the Thermomixer It is not necessary to close the tubes Caution Be careful when removing or adding liquid with the pipette from or to the AT do not touch the micro array at the bottom of the tube at any time Pipet all material in or out of the AT at the side of the bottom of the tube without touching the array as shown in the figure below S
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