User manual - GENOMICA SAU
Contents
1. 8 When visualizing the image in the reader confirm that position markers appear and that there are no bubbles or spots interfering with the reading If required clean the bottom of the tube with cellulose paper 6 SAMPLES BLOOD CULTURES The CLART SeptiBac kit has been designed and validated for its use in POSITIVE BLOOD CULTURES GENOMICA is not responsible for the results when using other samples The kit has been validated using both positive blood cultures with activated carbon and positive blood cultures with resin Blood cultures can be stored at 4 C for 24 h and at 20 C preferably at 80 C for an undefined period 7 WORKING PROTOCOL CLART SeptiBac Kit has been validated for automatic DNA extraction Performing a manual extraction procedures may affect the final result 7 1 Automatic DNA extraction 1 Sample preparation before extraction Recommended for all automatic equipments e Include a negative control in each samples serie to be performed The negative control should be a negative blood culture and should be processed like the rest of the samples e Mix by turning the blood culture Transfer 1 ml of blood culture to one 1 5 ml Eppendorf type tube and centrifuge for 2 min at 1500rpm Note Manipulations of blood cultures must be performed in biosafety cabinets using sterile material Take the supernatant into a sterile 1 5 ml tube and discard the pellet Note This step eliminates active carbo2. mix 1 tube Target specific oligonucleotides designed to detect microbial pathogens Mix 3 A pair of primers that amplify a fragment of human CFTR gene as a control for DNA extraction from the patient A pair of primers that amplify a modified plasmid included in the amplification tube and used as control in amplification of the PCR reaction e Specific oligonucleotides for the specific targets for detecting bacterial pathogens The reaction tube has been designed in order to favour the amplification of microorganisms comparing to the other two controls Among these two controls the amplification of the genomic DNA will be performed preferentially comparing to the control of the amplification reaction The reason for this design is Genomic DNA control would only be essential for confirming a negative result since it informs you that DNA from the patient was present in the sample even if no microorganism was found 19 Internal PCR control would only be essential when no amplification in the tube has been performed because it will help to distinguish between an inhibited PCR and a sample where no DNA is present There are two possibilities that may lead to a NOT VALID result e Not valid extraction The amplification of the microbial target and or the amplification of the extraction and amplification controls may be disturbed due to the presence of amplification inhibitors or due to a mechanical mistake during extraction Th
3. salts etc in the samples to be analyzed thus interfering with the genomic amplification and resulting in false negatives However the CLART SeptiBac eliminates false negatives by integrating different internal controls in the amplification tubes Mix1 Mix2 and Mix3 which are indicators of the amplification efficiency On top of that a genomic control extraction control has been included in Mix2 and Mix3 tubes avoiding false negatives due to a innefifcient extraction procedure 18 For the proper interpretation of the results the sample for detection of Gram positive bacteria and fungi must be processed in the amplification tubes Mix1 and Mix2 and visualized in Type 1 CS while the sample for detection of Gram negative bacteria must be processed in the amplification tubes Mix 3 and visualized in Type 2 CS Each amplification tube contains the following amplification oligonucleotides Mix 1 A pair of primers that amplify a modified plasmid included in the amplification tube and used as a control for amplification of the PCR reaction eTarget specific oligonucleotides designed to detect microbial pathogens Mix 2 A pair of primers that amplify a fragment of the human CFTR gene as a control for extraction of DNA from the patient A pair of primers that amplify a modified plasmid included in the amplification tube and used as a control for amplification of the PCR reaction This plasmid is different from that included in the
4. to the amplification tubes always in the cabinet During the process keep the tubes well separated and refrigerated 1 Thaw three reaction tubes per sample white green and blue on ice Do not use temperatures above 372C for thawing reagents 2 Centrifuge the reaction tubes in a microcentrifuge so that all the liquid goes to the bottom if no adapters are available to hold the reaction tubes they can be placed in larger tubes with their caps removed 3 Add 5 ul of extracted DNA from each sample to each amplification tube and resuspend several times with a micropipette Leave the tubes refrigerated at any time 5 Program the thermocycler as follows 14 1 cycle 952C 15 min 40 cycles 959C 15 sec 552C 15 sec 682C 45 sec 1 cycle 682C 10 min 49C maintained until tube collection optional 6 Start the program and place the amplification tubes in the thermocycler just when the block is above 909C This minimises any non specific amplifications due to hybridization occuring below the reaction temperature The amplification process lasts about 2 5 hours although this can vary depending on the thermocycler used Keep the amplified product at 4 C if used immediately or at 20 C if they will be used after several days 7 3 Visualization Protocol for CLART Strip CS Specific recommendations before starting the visualisation process THE PROTOCOL DESCRIBED BELOW SHOULD BE FOLLOWED IN THE POST PCR ARE
5. touching the bottom 9 Conserve revealed solution from light and not use after expiration date In If a precipitate appears product must be discarded VISUALIZATION SELECT THE ARRAY TYPE WHERE THE DIFFERENT AMPLIFICATION TUBES WILL BE VISUALIZED Type 1 CS Only intended for the visualization of the amplification products coming from tubes Mix1 white and Mix2 green providing results for the identification of Gram positive bacteria and fungi For the proper interpretation of the results it is necessary to visualize both tubes mixes 1 and 2 in the same well Type 2 CS Only intended for visualization of the amplification products coming from Mix3 tubes blue providing results for the identification of Gram negative bacteria For the proper interpretation of the results it is necessary to visualize the mix 3 tube 1 Denaturation use the thermocycler to denature the PCR products For this step place the already amplified tubes in the thermocycler and incubate at 95 C for 10 minutes Program the thermocycler for 15 minutes and remove the tubes after 10 minutes in order to keep them at 952C Remove the tubes from incubation at 95 C and place immediately in a container with ice It is recommended not to exceed 10 min denaturation time 2 Prepare TL diluted Solution For 8 wells one strip add 1 ml of TL solution to 9 ml of distilled water This will make up 10 ml of diluted TL solution necessary for one strip 3 P
6. 0 C 8 C Store between 4 9C and 8 2C 4C 18 C Store between 30 9C and 18 9C 30 C 2 DESCRIPTION CLART SeptiBac detects the presence of the following gram positive gram negative bacteria and fungi causing bacteriemia fungaemia sepsis or septic shock in positive blood cultures e Staphylococcus aureus e Staphylococcus epidermidis e Staphylococcus hominis e Staphylococcus haemolyticus e Streptococcus pyogenes dysgalactiae e Streptococcus pneumoniae e Streptococcus mitis e Streptococcus agalactiae e Streptococcus sanguinis parasanguinis e Streptococcus de grupo milleri including S constellatus y S anginosus e Detecci n gen rica para Streptococcus spp e Enterococcus faecium e Enterococcus faecalis e Gram positive cocci CGPs e Listeria monocytogenes e Clostridium perfringens e Escherichia coli e Klebsiella pneumoniae e Klebsiella oxytoca e Klebsiella spp K pneumoniae K oxytoca e Salmonella enterica e Enterobacter cloacae e Enterobacter aerogenes e Enterobacter spp E cloacae E aerogenes Citrobacter freundii e Citrobacter spp C freundii C koserii e Serratia marcescens plimutica e Proteus vulgaris penneri e Proteus mirabilis e Proteus spp P vulgaris P mirabilis P penneri e Haemophilus influenzae e Haemophilus spp H influenzae H parainfluenzae e Acinetobacter baumanii e Bacteroides fragilis e Bacteroides spp B fragilis B faecis e Pseudomo
7. A NEVER TAKE THE AMPLIFIED PRODUCT INTO THE PRE PCR AREA 1 Turn on the CAR CLINICAL ARRAY READER at the beginning of the process The calibration of the equipment takes a few minutes and samples references must also be entered in the program before reading The unit must be ready at the time of reading to avoid unnecessary delays that may lead to overexposed arrays 2 Switch on the thermomixer at 599C at least 60 min before starting the hybridization process 3 Allows the SH Hybridization Solution at room temperature 4 PREPARE THE WASHING SOLUTION BEFORE EACH RUN DO NOT USE PREVIOUS SOLUTIONS OR ANY REMAINING FROM PREVIOUS ASSAYS 5 Before starting the denaturing program wash the thermocycler with 1096diluted bleach During the denaturing process place the amplification tubes separated from each other into the thermocycler Do not denature for more than10 minutes 6 It is not necessary to use filter tips during the visualization process only when adding amplified products to every well However a different tip for each sample and for every reagent must be used This precaution must also be undertaken for the TL buffer 15 7 The 8 tip combs used with the aspiration pumps must be discarded after use or decontaminated with 10 bleach solution after each assay Make sure that the vacuum pump works properly and do not let remaining liquid in the wells 8 All the buffers must be thoroughly aspirated from the wells without
8. CLART GEnemicA CLART SeptiBac DETECTION AND MOLECULAR IDENTIFICATION OF BACTERIA AND FUNGI CAUSING SEPSIS FOR IN VITRO DIAGNOSTICS CLART SeptiBac CLART SeptiBac is under protection of patent family corresponding to International PCT Patent Application WO2015055768 CLART CLART Strip CAR SAICLART and SEPTIBAC are registered Trademarks of GENOMICA For further information and questions do not hesitate to visit www genomica com ud GENOMICA S A U Parque Empresarial Alvento Edificio B Calle V a de los Poblados 1 12 planta 28033 Madrid Spain www genomica com Version 5 July 2015 CONTENTS 1 GLOSSARY OF TERMS 2 DESCRIPTION 3 KIT COMPONENTS AND STORAGE 3 1 Amplification reagents 3 2 Visualization reagents 3 3 Other components 4 REQUIRED AND NOT SUPPLIED MATERIAL 4 1 Reagents and material 4 2 Equipment 5 HANDLING PROCEEDINGS AND RECOMMENDATIONS 5 1 General recommendations 5 2 Extraction precautions 5 3 Visualization precautions 6 SAMPLES BLOOD CULTURES 7 WORKING PROTOCOLS 7 1 Automatic extraction of genetic material 7 2 Amplification reaction 7 3 Visualization of amplified product 8 RESULTS READING 9 RESULTS INTERPRETING 10 TECHNICAL AND WORKING SPECIFICATIONS 11 BIBLIOGRAPHY 1 GLOSSARY OF TERMS Please check handling instructions Expiry date Sanitary product for n vitro diagnostics LoT Batch 25 C Store at room temperature 2
9. S with 200 ul of TL buffer at RT for 5 10 minutes or until the thermomixer has reached 25 C 17 It is very important that the diluted CJ buffer is completely washed off Any remaining buffer could react with the RE buffer producing an unspecific signal 8 Developing with RE buffer remove the diluted TL buffer without drying the array out and add 100 ul of RE buffer per well Incubate in the thermomixer at 25 C for 5 minutes without agitation Attention It is very important to use the thermomixer without agitation in this step 9 Remove the RE buffer with the pump The array must be dry at this time 10 CAR CLINICAL ARRAY READER Place the plate normally on the tray and the CAR will take and analyse the arrays automatically Firstly Type 1 CS Select software 1 CLART SeptiBac 1 for the interpretation of Gram positive bacteria and fungi results Secondly Type2 CS Select software 2 CLART SeptiBac 2 for the interpretation of Gram negative bacteria results 8 RESULTS READING The processing of the data obtained in each analysis is completely automatic The reading analysis equipment will provide a report with the results 9 RESULTS INTERPRETATION One of the main drawbacks of genomic amplification is the utilization of poor quality DNA samples too short DNA fragments degradation of DNA or loss of DNA during extraction or the presence of DNA polymerase inhibitors e g haemoglobin remains of paraffin wax
10. Table 1 Microorganism reaction 10 Enterobacter aerogenes 100 Citrobacter freundii 2 Salmonella enterica Staphylococcus epidermidis Candida glabrata 22 Candida spp Universal Hongos Escherichia coli 1000 000 Table 1 Number of copies of recombinant plasmid necessary to obtain 100 sensitivity in detecting each of the microorganisms Analytical specificity Several specificity experiments were conducted with 35 recombinant plasmids no nonspecific detection of other microorganisms other than that is to be determined was observed Therefore it can be considered that the technique achieves an almost 100 analytical specificity Diagnostic parameters In order to determine the diagnostic parameters of the kit CLART SeptiBac comparative studies against blood culture characterization were performed These comparisons were performed in collaboration with two Spanish hospitals and one Portuguese hospital Microbiology Service of the University Hospital Germans Trias i Pujol Badalona Barcelona Microbiology Service of the University Hospital Ramon y Cajal Madrid e Pathology and Laboratory Medicine of the Egas Moniz Hospital Lisbon Portugal Starting from blood cultures genetic material was extracted and analyzed for the presence of each of the microorganisms described in Table 2 We analyzed a total of 132 samples positive for Gram positive bacteria and fungi and 137 samples positive for Gra
11. e process has completely to be repeated e Not valid amplification The microbial presence in one tube and the absence of amplification in the other indicates a valid extraction procedure but indicates the presence of a problem during amplification The amplification with both tubes has to be repeated There are three possibilities that may lead to an UNCERTAIN result n those cases when replicates of an array probe are very different from each other n co infections of more than 3 bacteria for Mix 1 and Mix 3 and more than 2 microorganisms for Mix 2 When the signal intensity of non normalized absorbance is found at the detection limit of the technique which range is set by the software for each type of microorganism In cases of erroneous visualization of those amplification products in not specified CS according to the type of sample following possible interpretations should be considered Case 1 Visualization of an amplification product coming from tube mix 1 white in an Type 2 CS the result will be PCR inhibited Case 2 Visualization of an amplification product coming from tube mix 2 green in a Type 2 CS the result will be Negative in case of genomic control presence or PCR inhibited in case of no genomic control missed Case 3 Visualization of an amplification product coming from a tube mix 3 blue in an Type 1 CS the result will be Negative in case of in case of genomic control presence or PCR inhibit
12. ed in case of no genomic control missed 10 TECHNICAL AND WORKING SPECIFICATIONS 10 1 Known sources of interference 20 Certain substances can interfere with the CLART SeptiBac kit They are mainly substances inhibiting enzyme mixing and thus inhibiting the amplification reaction The most common factors are as follows Use of unsuitable samples The analysis of any kind of clinical samples other than those specified in the manual of the CLART SeptiBac kit as well as incorrect sampling can produce an inconclusive or invalid analysis result due to the lack of amplification as a consequence of sample shortage or inhibited reaction e Inadequate storage of samples can influence the result of the analysis If samples are subject to conditions that can result in the degradation of their DNA the result of the analysis might lead to a false negative e The presence of either hemoglobin active carbon or resins after extracting DNA might lead to PCR inhibitions 10 2 Technical specifications Analytical parameters e Analytical sensitivity Analytical sensitivity of microorganism presented in Table 1 was determined via amplification of a series of DNA dilutions of recombinant plasmids Each one of them contains the inserted amplified product including the complementary part of the detection specific probe The visualization step was performed in CLART Strip obtaining same results which are summarized in the following table
13. eptiBac detection system is based on the precipitation of an insoluble product in those areas of the microarray where hybridization of amplified products with specific probes occurs During the PCR the amplified products are labeled with biotin After amplification the products are hybridized to their respective specific probes that are immobilized at specific and known areas of the microarray These immobilised biotinilated products are recognized by the streptavidin of a streptavidin peroxidase conjugate thus providing with peroxidase activity to the hybridised products Peroxidase activity will then metabolise o Dianisidine and produce an insoluble product which will precipitate in those places where hybridisation occurred Fig 2 Probes on the array Biotin dit Incubation with conjugate gt Conjugate E GG gt Y 4 4 Spedfic precipitation Development reaction AV MY Za MY Figure 2 Diagram of the visualization method Probes immobilized on the surface capture their complementary biotin labeled amplified products With the help of the biotin the conjugate binds in this case streptavidin HRP HorseRadish Peroxidase Due to the HRP action the o dianisidine substrate produces a precipitation on the hybridization site 3 KIT COMPONENTS AND STORAGE CLART SeptiBac Kit contains enough reagents for the amplification and the analysis of 48 clinical samples The reagents are provided in two different boxes depend
14. erobacter cloacae 19 100 100 Enterobacter aerogenes 3 100 100 Salmonella 13 100 100 Citrobacter freundii 2 100 100 Citrobacter spp 1 100 100 Haemophylus influenzae 2 100 100 Haemophylus spp 1 100 Table 2 Diagnostic sensitivity and specificity of CLART SeptiBac for each microorganism PPV positive predictive value NPV negative predictive value Diagnostic reproducibilty and repeatability The obtained data are as follows For the detection of Gram positive bacteria and fungi e Reproducibility 95 4 n 44 samples e Repeatability 96 9 n 44 samples For the detection of Gram negative bacteria e Reproducibility 94 196 n 51 samples e Repeatability 98 6 n 49 samples Samples have been processed starting from the extraction step 23 1 11 BIBLIOGRAPHY Disefio y optimizaci n de un sistema de detecci n molecular por microarrays para la r pida identificaci n de bacterias grampositivas y hongos en hemocultivos positivos Moraga A l Salazar O Jordana LLuch E Martr E Molinos S Gim nez M Cospedal R Ausina V and M L Villahermosa Enfermedades Infecciosas y Microbiolog a Cl nica Vol 29 33 34 2011 15 Congreso de la Sociedad Espafiola de Microbiolog a Cl nica SEIMC Malaga 1 4 Junio 2011 Identification and characterization of bacterial pathogens and fungi causing blood infections sepsis by DNA microarrays Moraga A l Salazar O Manj n N Jo
15. eus P vulgaris P mirabilis P penneri Haemophilus H influenzae H parainfluenzae Acinetobacter baumanii Bacteroides B fragilis B faecis Pseudomonas P aeruginosa P putida P stuartii Stenotrophomonas maltophilia Extraction and amplification controls are included in this tube Note The kit package includes an adhesive and irreversible indicator of temperature the appearance of a reddish colour in the display window indicates that at some time products have exceeded the storage temperature of 209C and should not be used 3 2 Visualization reagents Visualization kit is shipped and should be storage at 49C WARNING Once received the kit the strips CLART Strip CS should be removed from the box and stored at room temperature provided that the temperature in the lab does not exceed 25 C CLART Strip CS including the specific probes These are delivered in a sealed envelope After opening the envelope should be closed and stored at room temperature protected from light exposure The kit contains two different strips identified as Type 1 CS and Type 2 CS Type 1 CS only intended for visualizing the amplification products coming from the tubes Mix1 white and Mix2 tubes green providing results for the identification of Gram positive bacteria and fungi For the proper interpretation of results both tubes must be amplified and visualized Type 2 CS intended only for the visualization
16. human flora therefore maximal precautions should be observed when extracting the genetic material from clinical samples Wear gloves at any time Gloves should completely cover the skin once disinfected without leaving the forearm exposed Put on gloves with fingers touching the edge of them Clean work surfaces with 1096 diluted bleach Turn on laminar flow and UV light at least 20 minutes before extraction The preparation of the samples before extraction should be performed inside the biosafety cabinet 5 3 Visualization precautions 1 Prevent the pipette tip or vacuum system from touching the bottom of the well since it might damage the microarray printed on it 2 It is recommended to add all solutions to the side wall of the CS never directly to the bottom 3 Add the hybridization solution SH immediately before adding the denaturalize PCR products 4 Using CS there will be a slight residual volume left avoiding the array to dry out 5 After incubating with the conjugate CJ it is very important to wash thoroughly the array in order to prevent any remaining residues from reacting unspecific with the developer RE thus generating an unspecific precipitate that might lead to misinterpretation of results 12 6 Avoid forming bubbles on the surface of the microarray when adding the different solutions 7 Keep the bottom of the CS clean in order to avoid possible interference when reading the results
17. ing on the temperature they should be stored at All the reagents provided are stable under the appropriate conditions until the indicated expiration date 3 1 Amplification reagents They are delivered and should be storage at 209C Amplification tubes are provided ready to use They contain 45 ul of reaction mix Only the required number should be thawed on ice at any given time while the remaininds should be kept at 202C Three different amplification tubes are included in the kit Mix 1 White tube Amplification of Staphylococcus aureus Staphylococcus epidermidis Staphylococcus hominis Staphylococcus haemolyticus Streptococcus pyogenes dysgalactiae Streptococcus pneumoniae Streptococcus mitis Streptococcus agalactiae Streptococcus sanguinis parasanguinis Streptococcus milleri group including S constellatus and S anginosus Streptococcus spp Enterococcus faecium Enterococcus faecalis Listeria monocytogenes and mecA Amplification control is included Mix 2 Green tube Amplification of Clostridium perfringens Candida albicans Candida glabrata Candida krusei Candida spp and generic marker for detecting fungi Extraction and amplification controls are included in this tube Mix 3 Blue tube Amplification of Escherichia coli Klebsiella K pneumoniae K oxytoca Salmonella enterica Enterobacter E cloacae E aerogenes Citrobacter C freundii C koserii Serratia S marcescens S plimutica Prot
18. ler e Biosafety cabinet type Il eThree adjustable micropipettes 1 20 ul 20 200 ul and 200 1000 ul for use in the extraction area e One adjustable micropipette 1 20 ul for adding extracted products in to amplification tubes eThree adjustable micropipettes 1 20 ul 20 200 ul and 200 1000 ul for use in the visualization laboratory 10 Thermoblocks at 25 C 30 C and 592C with an adjustable agitation source and compatible with microtitter plates Vortex e Vacuum system e Automatic extractor unit 5 HANDLING PROCEEDINGS AND RECOMMENDATIONS Very important in order to avoid potential contaminations read this section carefully before beginning any work 5 1 General recommendations 1 The procedure should be performed in two physically separated areas This will avoid contamination of samples with previously amplified products Each area should have its own identified working materials pipettes tips tubes racks gloves etc which should never leave the assigned area 1 Pre PCR area This area is dedicated to DNA extraction and sample preparation This preparation must be done within an adequate biosafety cabinet and observing the broader sterilization measures possible to avoid potential contamination 2 Post PCR area This area is dedicated to carry out amplification and visualization of the amplified product The material in this area has never come into contact with the extraction area Avoid going t
19. m negative bacteria It should be noted that samples with coinfections were found so that the total amount of microorganisms analyzed is higher than the number of samples analyzed CLART SeptiBac results were compared to the results of the reference technique Blood culture characterization In case of discrepancies the results of sequencing were considered as the valid results In those specific cases where this information was not available the discrepancies were evaluated by Nested PCR and further DNA sequencing e Diagnostic specificity 23 The technique has also been validated with negative blood cultures co infection gram negative and gram positive and with the presence of other organisms whose detection is not covered by the kit The results showed no cross reactivity Staphylococcus aureus 16 100 Staphylococcus epidermidis 45 icus 3 100 100 Streptococcus 100 9 6 8 95 6 94 1 100 pyogenes dysgalactiae 5 Streptococcus agalactiae 6 Streptococcus mitis 2 Streptococcus sanguinis parasanguinis 5 Streptococcus grupo milleri 1 S constellatus y S anginosus EIE LI enorme Deep EE mw 8 9 Bacteroides fragilis Pseudomonas spp 32 Bacteroides spp 5 1 E coli 45 Serratia 7 5 4 8 iae 2 100 5 6 100 6 7 6 2 100 100 100 100 100 100 8 9 100 100 100 100 100 3 8 100 100 100 100 75 100 100 100 00 1 24 Ent
20. n excess and or resins present in the blood culture It is sometimes difficult to visualize the pellet and therefore the supernatant should be taken carefully 13 Centrifuge the supernatant at 13000rpm for 5 min to concentrate the bacterial and fungal cells that might be in suspension Discard supernatant and resuspend in 1 ml of saline solution sodium chloride 0 9 Resuspend vigorously Transfer 1 ml to the extractor Sometimes the activated carbon and resins create agglomerates Do not transfer these agglomerates to the extractor since they might obstruct the extractor pipettes 2 Internal lysis and DNA extraction in NucliSENS easyMAG equipment from Biom ri ux follow the computer s user guide The elution volume selected is 80 Bl This volume may vary depending on the automatic extractor unit used 3 Once the extraction is made transfer 80 AO of eluted DNA with the micropipette into an Eppendorf tubes of 1 5 ml Use 5 ul for every tube of amplification and store the rest at 20 C It is important to include an extraction negative control to verify that the samples have not been contaminated during the extraction amplification or visualization processes thus giving rise to a false positive 7 2 Amplification reaction Specific recommendations for amplification e Work in pre PCR area always using cabinets and following the recommentations described in paragraph 5 1 e Add the DNA
21. nas spp P aeruginosa P putida P stuartii e Stenotrophomonas maltophilia e Candida albicans e Candida glabrata e Candida krusei e Generic detection for Candida spp detection of Candida parapsilosis not included and fungal taxa Including the detection of mecA region responsible of the appearance of methicillin resistance in Staphylococci genus Detection of the different microorganisms is achieved by PCR amplification of a specific fragment ranging 149 500 bp size Amplification is performed in three different types of PCR tubes Mix 1 tubes are white and allow the amplification and subsequent detection of Gram positive bacteria excluding Clostridium perfringens Mix 2 tubes are green and contain everything needed for amplification of fungi and Clostridium perfringens Mix 3 tubes are blue and contain everything needed for the amplification and subsequent detection of Gram negative bacteria The detection of the product amplified by PCR is carried out by means of a low density microarray platform CLART Clinical Arrays Technology The platform is based on a very simple principle but at the same time cost effective It consists of a microarray printed at the bottom of a microtiter plate which simplifies the entire hybridization and visualization process when compared to classic microarray systems Figure 1 displays a CLART Strip or CS of 8 wells Figure 1 CLART Strip in 8 well strip format CS The CLART S
22. o the pre PCR area after having worked in the area of post PCR 2 Use gloves at all times It is recommended to change gloves quite frequently and it is mandatory to change them prior to start working in each of the above mentioned areas New gloves should be used for the preparation of the amplification tubes and every time DNA is added to them The user working in the pre PCR area should use skin disinfectant solutions to prevent contamination from epidermal flora In addition gloves should completely cover the skin once disinfected without leaving the forearm exposed 3 Clean working areas laboratory benches hoods grids pipettes thermocycler thoroughly with 10 diluted disinfectant following every sample batch processing it is mandatory to disinfect all working areas preventing contaminations 4 Always use pipette tips containing a filter or use positive displacement pipettes to avoid contamination 11 5 Use disposable and autoclaved laboratory materials 6 Do not mix reagents from two different tubes even though they belong to the same lot 7 Close reagent tubes immediately after use as this will avoid contamination 8 Dispose of micropipette tips after use 9 GENOMICA does not assume any responsibility for those results obtained when performing other samples different from those mentioned 5 2 Extraction precautions Many of the microorganisms in CLART SeptiBac kit are part of the normal physiological
23. of the amplification product derived from Mix3 tubes blue providing results for the identification of Gram negative bacteria SH Hybridization solution Store at 49C e DC Conjugate diluent Store at 49C CJ Conjugate Store at 49C Centrifuge briefly before using RE Developer Store at 42C and protected from light e TL Washing buffer Store at 42C Support and lid for the 8 well strip 3 3 Other components e CAR CLINICAL ARRAY READER which allows the reading and automatic interpretation up to 12 CS which means a total amount of 96 samples This platform is manufactured exclusively for GENOMICA kits use only SAICLART software developed by GENOMICA for image processing e CLART SeptiBac Software It is specific for CLART SeptiBac designed and validated by GENOMICA for each specific array that composes the kit Software 1 To be used exclusively for the analysis of Type 1 CS Gram positive bacteria and fungi Software 2 To be used exclusively for the analysis of Type 2 CS Gram negative bacteria Figure 3 CAR reader 4 MATERIAL REQUIRED AND NOT PROVIDED 4 1 Reagents and materials Distilled water Disposable gloves Positive displacement or filtered pipette tips Bowl of chipped ice e Sterile Eppendorf type tubes 1 5 ml Racks for 1 5 ml tubes Racks for 0 5 ml 0 2 ml tubes e Saline buffer NaCl 0 9 4 2 Equipments Microcentrifuge e Thermocyc
24. quicker 5 Double washing Wash the wells twice by adding 200 ul of diluted TL Solution and mix it 10 to 15 times with the pipette Remove the diluted TL Solution using a pipette or a vacuum system without leaving any remanent volume Repeat the process This step must be carried out with different tips for each well in both washed In case the heating block has not reached a temperature of 309C when you get to this step leave the CSs filled with diluted TL Solution until the heating block reaches the necessary temperature 6 Blocking and conjugate It is recommended to spin the CJ buffer for 10 seconds before using Prepare the diluted CJ solution 15 minutes before hybridization time is over and keep it on ice until its use For one strip 8 wells adds 7 5 ul of high affinity CJ buffer to 1 ml of DC buffer Remove the diluted TL buffer avoiding the array to dry out and add 100 ul of diluted CJ buffer to each well Incubate in the thermomixer at 30 C 550 rpm for 10 minutes exactly After this incubation take the strip and remove the diluted CJ buffer immediately with the pump Set the thermomixer at 25 C and no shaking for step 8 Remove the lid to speed up the cooling 7 Triple Washing Remove the diluted CJ buffer and add straight away 200 pl of TL diluted solution per well Mix it 10 to 15 times with the pipette and remove the diluted TL buffer with the pump without drying the array out Repeat this washing twice and leave the C
25. rdana E Martr E Gim nez M Cospedal R Ausina V and M L Villahermosa Proceedings of the 22 European Congress of Clinical Microbiology and Infectious Diseases ECCMID 2012 London UK March 30 April 2 2012 Disefio y optimizaci n de un sistema de detecci n molecular por microarrays para la r pida identificaci n de bacterias gram negativas en hemocultivos positivos Salazar Torres O Manjon Vega N Moraga Quintanilla A l Jordana Lluch E Martro Catala E Molinos Abos S Gimenez Perez M Cospedal Garcia R Ausina Ruiz V Villahermosa Jaen M L Congreso de la Sociedad Espafiola de Microbiologia Cl nica SEIMC Bilbap 9 11 Mayo 2012 26
26. re washing of CS Add 200 ul of diluted TL Solution to every well and mix it up 10 to 15 times with a multichannel pipette avoiding the contact with the array It is recommended to prewash the strips while denaturation process is performed keeping the arrays on washing buffer until adding the denaturate samples Remove the diluted TL Solution using a pipette or preferably a vacuum system 16 The CS should not contain washing solution residues Under no circumstances should CSs be allowed to dry out for a long period of time 4 Hybridization Hybridization solution SH must be heated at 592C in order to dissolve crystallized salts Add 100 ul of SH buffer avoiding the appearance of foam on each well For each array add a Type 1 CS add 5 ul of the denatured PCR product from both mix 1 white tube and mix 2 green tube b Type 2 CS add 5 ul of the denatured PCR product from mix 3 blue tube Mix properly with the pipette avoiding touching the array and incubate the strip covered with the transparent plastic lid in the thermomixer for 30 minutes at 59 C shaking at 550 rpm We recommend adding the amplified products on each strip independently and separately from the rest to avoid contaminations Remove the CSs and remove the SH Solution using a pipette or a vacuum system Program the heating block at 309C and leave it running so that it can be used later on step 6 You can remove the lid from the heating block to let it cool down
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