User manual - GENOMICA SAU
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1. e Adaptor and lid for 8 well strips 3 4 Other components The following components are required for the capture and subsequent image processing e CAR CLINICAL ARRAY READER which allows the reading and automatic interpretation up to 12 CS that means a total amount of 96 samples This platform is manufactured exclusively for GENOMICA kits use only e SAICLART software developed by GENOMICA for image processing e CLART PneumoVir Software It is specific for CLART PneumoVir designed and validated by GENOMICA Installed and ready to use Figure 3 CAR CLINICAL ARRAY READER 4 MATERIALS REQUIRED BUT NOT PROVIDED Below you can find a list of all materials required but not provided 4 1 Reagents and materials Distilled water Saline solution Disposable gloves Filter tips or positive displacement pipettes Crushed ice container 1 5 ml autoclaved Eppendorf type tubes 1 5 ml tube grids 0 5 ml 0 2 ml tube holder 4 2 Equipment autoclart Figure 4 The following equipment is needed for the automatic visualization phase It enables the automatic visualization of up to 12 CS that means a total amount of 96 samples Figure 4 autoclart e Microcentrifuge e Thermocycler e Laminar flow chamber for the extraction laboratory e Three adjustable micropipettes ranging from 1 20 ul 20 200 ul and 200 1000 ul for the extraction laboratory e One adjustable micropipette2. 83 33 99 63 87 50 99 63 100 00 100 00 96 55 100 95 100 100 00 100 00 100 00 100 00 Table 2 Diagnostic sensitivity and specificity of CLART PneumoVir for each virus 22 11 BIBLIOGRAPHY Heyman PVV Carper HT Murphy DD Platss Mills TA Patrie J McLaughlin AP Viral infections in relation to age atopy and season of admission among children hospitalized for wheezing J Allergy Clin Immunol 2004 114 239 47 Spicuzza L Spicuzza A La Rosa M Polosa R Di Maria G New and emerging infectious diseases Allergy Asthma Proc 2007 28 1 28 34 Boschini A Longo B Caselli F Begnini M De Maria C Ansaldi F Durando P Icardi G Rezza G An outbreak of influenza in a residential drug rehabilitation community J med Virol 2006 78 9 1218 22 Herrera GA Iwane MK Cortese M Brown C Gershman K Shupe A Averhoff F Chaves SS Gargiullo P Bridges CB Influenza vaccine effectiveness among 50 64 year old persons during a season of poor antigenic match between vaccine and circulating influenza virus strains Colorado United States 2003 2004 Vaccine 2007 Jan 2 25 1 154 60 Hammond S Chenever E Durbin JE Respiratory virus infection in infants and children Pediatr Dev Pathol 2007 May Jun 10 3 172 80 Marta Cruz Ca ete David Moreno P rez Antonio Jurado Ortiz Francisco Jesus Garcia Martin Juan L pez Siles Laura Olalla Martin Enferm Infecc Microbiol Clin 2007 25 177 183 M A
3. Marcos M Camps J Puig de la Bellacasa T Pumarola E Garcia J Mensa A Torres y M T Jim nez de Anta Enferm Infecc Microbiol Clin 2004 22 40 46 Vicente D Human bocavirus a respiratory and enteric virus Emerg Infect Dis 2007 Apr 13 4 636 7 Coiras MT Aguilar JC Garcia ML Casas Perez Brena P Simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested PCR assays J Med Virol 2004 Mar 72 3 484 95 Coiras MT Perez Brena P Garcia ML Casas Simultaneous detection of influenza A B and C viruses respiratory syncytial virus and adenoviruses in clinical samples by multiplex reverse transcription nested PCR assay J Med Virol 2003 Jan 69 1 132 44 Elliot A J Cross K W Fleming D M Acute respiratory infections and winter pressures on hospital admissions in England and Wales 1990 2005 J Public Health Oxf 30 91 9 Cannon J A Carr M J Yandle Schaffer K Kidnay R Hosny G Doyle G Ryan J Gunson R Collins T Carman W F Conell F and W Hall A low density oligonucleotide microarray for the detection of viral and atypical bacterial respiratory pathogens Journal of Virological 23 Methods Volume 163 Issue 1 January 2010 Pages 17 24 Tract Viral infections and Coinfections in Patients with Influenza like Illnesses by use of RT PCR DNA Microarray Systems J Clin Microbiol doi 10 1128 JC
4. 1 Use globes Clean working surfaces of cabinets with a 10 diluted bleach solution 3 Turn on the laminar flow and UV light at least 20 minutes before extraction Turn off the UV light when it is working inside the cabinet 4 The preparation of the samples before extraction must be made inside the cabinet ha 5 3 Precautions for amplification e Place the amplification tubes in the thermocycler when the block is above 90 2 C Thereby minimizing possible nonspecific amplifications due to incubation below the annealing temperature 5 4 Precautions for visualization 1 Avoid the pipette tip or the vacuum system touching the bottom of the tube since this could damage the microarray 2 It is recommended to add all solutions to the wall side of the CS never directly to the bottom 3 It is advisable not to add the SH solution until the addition of the denatured PCR products 4 Following incubation with the CJ solution it is very important to wash the CS thoroughly to avoid any residues that could react with the RE solution resulting in a non specific precipitation that could lead to false result interpretations 5 Avoid bubbles on the microarray surface when adding any solution 11 6 Clean the back of the CS externally to avoid possible interferences during the results reading 7 When visualizing the image in the reader confirm that position markers appear and that there are no bubbles or spots interfering with the
5. in the pre PCR area for the amplification tubes preparation always using a hood and following the recommendations mentioned in section 5 1 e Be very careful when adding the enzyme mixture since it contains a high percentage of glycerol This way if you introduce the pipette tip too deep the mixture adheres to the walls causing the addition to the reaction tube of a larger amount of mixture than the necessary and some loss of enzyme volume this could result in an insufficient enzyme volume for the rest of the amplification tubes of the kit e Add the extracted DNA RNA in the pre PCR area always working under the hood and following the recommendations mentioned in section 5 2 During the process keep tubes separate and on ice Amplification reaction protocol 13 1 For each sample to be processed thaw and keep 2 amplification tubes a colorless and green one on ice 2 Centrifuge the reaction tubes in the microcentrifuge for a few seconds so that all liquid can accumulate at the bottom of the tube In case there are no microcentrifuge adaptors available for reaction tubes larger tubes can be used instead after having cut their cap off 3 Add 2 pl of the enzyme mixture in both colorless and green tubes 4 Add 5 pl of the extracted RNA DNA to both reaction tubes and mix several times with the micropipette Keep the tubes on ice 5 Program the following temperature cycles on the Thermocycler 1 cycle 452C 45 min 952C 15 m
6. ranging from 1 20 ul to add the enzyme mixture to the amplification tubes e One adjustable micropipette ranging from 1 20 ul to add the genetic material to the amplification tubes e Three adjustable micropipettes ranging from 1 20 ul 20 200 ul and 200 1000 ul for the visualization laboratory e Heating block with agitation adjustable temperatures 252C 302C 502C 532C and 5920 Interchangeable blocks compatible with 1 5 ml Eppendorf Tubes and 96 Wells Microtiter Plate e Vortex e Vacuum system 5 RECOMMENDATIONS AND HANDLING PROCEDURES Very important in order to avoid contamination Read carefully before initiating the assay 5 1 General recommendations 1 This assay should be performed in FOUR physically separated AREAS in order to avoid sample contamination with the previously amplified product Separate working materials should be available in each area pipettes tips tubes grids gloves etc which should never be used outside these areas e Pre PCR extraction area DNA RNA extraction occurs in this area A laminar flow hood should be used e Pre PCR area for the preparation of the amplification tubes In this area the enzyme mixture is added to the amplification tubes It is recommended to use a laminar flow hood e Pre PCR area for the addition of the extracted material In this area the extracted DNA RNA is added to the amplification tubes where the enzyme mixture has been previously introduced A laminar fl
7. reading You may clean the bottom of the tube with cellulose paper or gently tap the tube with your finger 6 SAMPLING The CLART PneumoVir kit has been designed and validated to be used with DNA extracted from respiratory samples nasopharyngeal lavage and exudated pharyngeal exudated and broncoalveolar lavage BAL GENOMICA is not responsible for the results obtained if other types of samples are used Store samples at 4 C if they are to be processed in a time less than 12h Otherwise it should be stored frozen at 20 or 80 C 7 WORKING PROTOCOL An incorrect performance during the DNA RNA extraction procedure may lead to false negative results Especial attention at this step is highly recommended In order to optimize results a minimal amount of 5 10 ng ul DNA RNA is required as extraction output independently if it is performed manually or automatically 7 1 Manual Extraction of DNA RNA of different clinical samples Specific recommendations before initiating extraction Work in the pre PCR extraction area always using a laminar flow hood and following the recommendations mentioned in section 5 1 and 5 2 Keep samples on ice and well separated Add reagents in the indicated order Do not use saline solution for swabs Extraction protocol 1 Include a negative control in each sample batch consisting of 200 ul of RNA free water and process like the rest of the samples 2 Pipette 200 ul of clinic
8. specific container autoclart calculates the specific volumes required according to the amount of samples indicated TL Washing buffer Volume showed in the display indicates the diluted washing buffer required In order to prepare the diluted washing buffer please dilute the TL reagent provided 1 10 into distilled water SH Hybridization solution It is provided ready to use Add the specified volume in the container once tempered CJ Conjugate It s recommended to shortly spin the CJ before use Display shows final volume of diluted CJ meaning that each ml indicated on the display should be prepared as follows 1 ml of DC Conjugate Diluent and 5 ul CJ reagent Vortex the diluted solution in order to mix it properly up RE Developer Add the RE volume indicated on the display 13 Close the door and press the knob The device will start priming the system and cleaning the tips with water Then it will perform the pre washes of the CS and adding the hybridization solution Once finished these steps the device will beep as a signal for pipetting the samples on their specific CS autoclart will automatically stop beeping as soon the user opens the door 14 For adding the samples on the CSs please remove the plate carefully from autoclart unit and add 3 ul of each denatured amplification tube colorless and green one to the CLART Strip Resuspend several times to mix with the SH Solution without touching the array
9. CLART GEnemicA CLART PneumoVir CHARACTERIZATION OF VIRUSES CAUSING HUMAN RESPIRATORY INFECTIONS VIA GENOMIC IDENTIFICATION FOR JN VITRO DIAGNOSIS CLART PneumoVir CLART PneumoVir is under protection of patent family corresponding to International PCT Patent Application WO2009144497 which comprises national and regional members in different territories including granted patents in Europe Mexico and Russia and patent applications under prosecution in Brazil and Canada CLART CLART Strip CAR SAICLART AUTOCLART and PNEUMOVIR are registered Trademarks of GENOMICA For more information please refer to the web site www genomica com ial GENOMICA S A U Parque Empresarial Alvento Edificio B Calle Via de los Poblados 1 12 planta 28033 Madrid Spain www genomica com Version 11 July 2015 TABLE OF CONTENTS 1 KEY TO SYMBOLS 2 PROTOCOL DESCRIPTION 3 KIT COMPONENTS AND STORAGE 4 1 Extraction reagents 4 2 Amplification reagents 4 3 Visualization reagents 4 4 Other components 4 MATERIALS REQUIRED NOT PROVIDED 4 1 Reagents and materials 4 2 Equipment 5 RECOMMENDATIONS AND HANDLING PROCEDURES 5 1 General recommendations 5 2 Precautions for the extraction and addition of extracted material to the amplification tube 5 3 Precautions for amplification 5 4 Precautions for visualization 6 SAMPLING 7 WORKING PROTOCOL 7 1 Extraction of genetic material of viruses associated
10. M 00733 10 Characterization of viruses causing Human Respiratory Infections via Genomic Identification for in vitro diagnosis CLINICAL ARRAYS CLART PneumoVir Poster Session at ECCMID 2008 Barcelona Myocarditis Caused by Human Parainfluenza Virus in an Immunocompetent Child Initially Associated with 2009 Influenza A H1N1 Virus Journal of Clinical Microbiology May 2011 p 2072 2073 Respiratory viruses in Children Admitted to Hospital Intensive Care Units Evaluating the CLART PneumoVir DNA Array Journal of Medical Virology 83 150 155 2011 Evaluation of viral co infections in hospitalized and non hospitalized children with respiratory infections using microarrays Clin Microbiol Infect 10 1111 1469 0691 12015 Co infeccion viral respiratoria en ni os hospitalizados por infecci n respiratoria aguda y su impacto en la gravedad cl nica Rev Chil Infect 2012 29 2 169 174 Broad Respiratory Virus Detection in Infants Hospitalized for Bronchiolitis by Use of a Multiplex RT PCR DNA Microarray System Journal of Medical Virology 84 979 985 2012 Detection and genotyping of human respiratory viruses in clinical specimens from children with acute respiratory tract infections Rev Esp Quimioter 2013 26 1 47 50 24
11. Place the microplate again on the autoclart Press the knob again to continue the visualization process 15 Once finished the visualization process the autoclart unit will beep again indicating the end of the run Please remove the microplate carefully and proceed with the reading step on the CAR 16 CAR CLINICAL ARRAY READER place the plate normally on the tray and the CAR will take and analyse the arrays automatically 8 RESULTS READING Processing of data obtained from each analysis is carried out automatically The reading and analysis system will present a report indicating the results The system monitor displays a three column table the left column shows the virus species and the subtypes characterized in the micro array The central column shows a positive or negative result for each virus species while the right one shows the validity determined by the DNA RNA extraction and amplification control 9 RESULTS INTERPRETATION 17 One of the main drawbacks of the detection by genomic amplification are false negatives which are mainly due to the presence of inhibitors of the enzyme mixture RT and DNA polymerase in those samples where virus detection is going to be performed hemoglobin salts etc The CLART PneumovVir kit eliminates such false negatives thanks to the addition of an internal control of the amplification reaction efficiency in the amplification tubes An incorrect performance during the DNA RNA extr
12. action procedure may lead to false negative results Especial attention at this step is highly recommended In every set of analysis a negative extraction control should be included to check that samples have not been contaminated during the extraction amplification and visualization thus giving rise to a false positive When the viruses are present in the sample there is always a preference to amplify the viral genotypes instead of amplifying the controls Hence under certain conditions i e high number copies of one particular virus genotype or when several genotypes are present in the samples internal controls may not appear No signal Considering all these information we could interpret the reading results as 1 Positive Samples 1 1 With positive amplification control Virus Result Control Species Positive Passed Control Signal Result Internal Control gt 0 165 Passed e CLART Strip CS Amplification Control Alignment dots Internal Control Virus This is a VALID RESULT Result can be considered as a real positive 1 2 With negative amplification control Virus Result Control Specie Positive Passed 18 Control Signal Result Internal Control lt 0 165 No Signal e CLART Strip CS Strip Alignment dots Virus Even though the Internal Control is not appearing results can be considered as valid This is due to the
13. al sample In case of swabs with transport media vortex for 30 seconds and then pipet 200 ul 12 3 Add 600 ul of SEML liquid sample extraction solution Wait until the solution thaws and turns clear before using it Mix by inverting the tubes several times and allow 15 min at room temperature 4 Add 600 ul of isopropanol stored at 202C mix by inverting the tubes several times and centrifuge preferably at 42C at 13 000 rpm for 20 min 5 Remove supernatant using a micropipette A 1000 ul micropipette can be used to remove the supernatant as long as a smaller micropipette is used at the end for example a 20 ul one for removing the residues at the bottom of the tube without removing the precipitate by mistake 6 Add 1000 ul of 70 ethanol stored at 202C Agitate gently to clean the precipitate at the bottom 7 Centrifuge preferably at 42C at 13 000 rpm for 15 min 8 Remove the supernatant carefully as indicated at step 4 Allow the sample to dry under the hood for 15 or 20 min until there are no ethanol residues left Prior to resuspending the sample confirm that there are no ethanol residues left 9 Resuspend in 20 ul of Dilution solution 7 2 Automatic Extraction Please follow the recommendations and protocol provided by the extractor supplier and check if the extracted material fulfills CLART PneumoVir protocol s requirements 7 3 RT PCR amplification Amplification specific recommendations e Work
14. apsed amplified products can continue at 952C Remove the tubes from the 952C incubation and place them immediately in a container with ice 2 Diluted TL Solution preparation Prepare 10 mL strip of fresh TL solution by diluting 1mL TL into 9 mL distilled water 3 CS preliminary washing Add 200 ul of diluted TL Solution to every array and invert the tube 10 to 15 times Discard the diluted TL Solution using a pipette or preferably a vacuum system This step is necessary in order to wash already packaged tubes before adding the sample The tube should not contain washing solution residues for this reason residual volumes in the caps should be aspirated with a vacuum system Under no circumstances should tubes be allowed to dry out for a long period of time 4 Hybridization Once PCR products have been denatured add 100 ul of tempered SH solution in each well avoiding foaming Add 3 ul of each denatured amplification tube colorless and green one to the CLART Strip Resuspend several times to mix with the SH Solution without touching the array Incubate in the Thermoblock for 1 hour at 592C agitating at 550 rpm Following incubation remove the tubes and discard the SH Solution using a pipette or a vacuum system Program the Thermoblock at 302C and leave it running so that it can be used later on step 6 You can remove the lid from the Thermoblock so that it can cool down quicker 5 Washing Wash every well of the CS twice with 200
15. competition among different targets during the amplification process It is a REAL POSITIVE RESULT 2 Negative samples Virus Result Control Specie Negative Passed Control Signal Result Internal Control gt 0 165 OK s CLART Strip CS Amplification Control Alignment dots Internal Control It is considered as a VALID RESULT In this case result can be considered as a REAL NEGATIVE 3 Inadequate samples inhibited Virus Result Control Specie Negative PCR Inhibited 19 e R CLART Strip CS Alignment dots It is considered as an INVALID RESULT Amplification process has been interfered by an unknown substance which might inhibit the DNA polymerase enzyme At this point is recommended to verify the presence of any PCR inhibitor substance in the sample or the extracted material If so please proceed to extract the sample again or ask the doctor to repeat the sampling process There are three possibilities of obtaining an Uncertain Result e Those cases where the three virus replicas are very different from each other shape and intensities e In co infections with more than 5 virus e When the raw absorbance signal is in the range established as uncertain for each virus type 10 TECHNICAL AND OPERATIONAL SPECIFICATIONS 10 1 Control of known interferences False negatives are one of the drawbacks in the detection by genomic amplification due to eithe
16. in 45 cycles 952C 0 5 min 502C 1 5 min 682C 1 0 min 1 cycle 682C 10 min 42C continuously until tube collection optional The amplification lasts around 5 hours although this could slightly vary depending on the Thermocycler 7 4 Amplified product visualization on CLART Strip CS Specific recommendations 1 THE BELOW DESCRIBED PROTOCOL SHOULD ALWAYS BE USED IN THE POST PCR AREA NEVER TAKE THE AMPLIFIED PRODUCT IN THE PRE PCR AREA 2 Ensure that the Thermomixer has been at 59 2C at least 60 min before starting the incubation time 3 Keep the SH solution in the thermomixer at 592C hybridizing temperature 4 Prepare the washing solution before each assay Do not use previous solutions or any remaining from previous assays 5 It is not necessary to use filter tips during the visualization process However a different tip for each sample and for every reagent must be used 6 The 8 tip combs used with the aspiration pumps must be cleaned after use or decontaminated with 10 bleach solution after each assay Please ensure that the vacuum pump works properly and does not left remaining volumes on the well 14 7 Aspirate the different solutions completely without touching the array 7 4 1 Manual visualization 1 Denaturation use the Thermocycler to denature the amplified products For this step place the tubes in the Thermocycler and incubate at 952C for 8 min Program 10 minutes so that after 8 minutes have el
17. oper performance of the amplification process 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 on a microarray printed at the bottom of a microtiter plate well CLART Strip CS Figure 1 which simplifies the entire hybridization and visualization process when compared to classic microarray systems Figure 1 CLART Strip CS platform in the form of an 8 well strip CLART PneumoVir detection system is based on the precipitation of an insoluble product at those sites of the microarray where the hybridization of the amplified products by specific probes takes place During RT PCR amplified products are labeled with biotin After amplification they hybridize with their respective specific probes that are immobilized on the array and then incubated with a streptavidin peroxidase conjugate The conjugate binds via streptavidin with the biotin present in the amplified products which are also bound to their specific probes while in the presence of o dianisidine the peroxidase activity of the conjugate induces the appearance of an insoluble product which precipitates at the hybridization sites of the microarray Figure 2 Labelled products hee E Conjugate 2 NS rs a NA A Development reaction F aa Figu
18. ow hood should be used e Post PCR area Amplification and visualization of the amplified product are carried out in this area 2 Always use gloves It is recommended to change gloves quite frequently while 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 RNA is added to them 3 Clean working areas laboratory benches hoods grids pipettes thoroughly with 10 diluted bleach following every sample batch processing it is mandatory to disinfect all working areas in case of contamination When using Thermoshaker or Themocycler it s highly recommended to clean prior and after their use 10 4 Always use filter tips and positive displacement pipettes to avoid contamination due to micropipettes A different set of pipettes should be used in each area 5 Discard the micropipette tip after pipetting 6 Use disposable and autoclaved laboratory material 7 Never mix reagents from two different tubes even if they belong to the same lot 8 Close reagent tubes immediately after use in order to avoid contamination 9 GENOMICA is not responsible for results obtained using this kit in case of use of samples other than those indicated or DNA RNA extracted with a protocol other than the one indicated herein 5 2 Precautions for the extraction and addition of extracted material to the amplification tube
19. r an inadequate quality of the extracted DNA due to insufficient sample quantity DNA degradation inadequate storage or DNA loss during extraction or to the presence of DNA polymerase inhibitors in the samples that are to be processed alcohol salts etc To avoid these interferences the indications appearing in the sections 5 6 and 7 of this manual must be followed 10 2 Technical specifications Processing parameters 20 Analytical sensitivity Analytical sensitivity of the virus types 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 results which are summarized in the following table Viruses associated with respiratory infections Recombinant plasmid copy N2 per PCR reaction Metapneumovirus Coronavirus Influenza virus A human H1N1 human H3N2 Influenza A H1N1 2009 Influenza virus B Influenza virus C 100 Parainfluenza virus 4 RSV A RSV B Adenovirus Bocavirus Enterovirus Echovirus Parainfluenza virus 1 Parainfluenza virus 2 1000 Parainfluenza virus 3 Rhinovirus Table 1 Relation between copy numbers of the recombinant plasmid specified by virus type necessary for obtaining a 100 sensitivity in the detection of each virus e Analytical
20. re 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 Thanks to the HRP action the o dianisidine substrate produces a precipitation on the hybridization site 3 KIT COMPONENTS AND STORAGE CLART PneumoVir kit contains sufficient reagents for the extraction and analysis of 16 48 or 96 clinical samples Reagents included in the kit have been grouped in various packages depending on the temperature they should be stored at When storage recommendations are observed all reagents remain stable until the kit expiration date 3 1 Extraction reagents The extraction purification kit is shipped at 202C and it should be stored at this temperature until its use e SEML extraction solution Once thawed it should be stored at 42C and used within 8 days e SD Dilution solution Store at 202 or 42C e IP lsopropanol Store at 208C e DE 70 Ethanol Store at 202C 3 2 Amplification reagents They are shipped and stored at 202C e Ready to use amplification tubes They contain 43 uL of reaction mixture Only thaw on ice the exact number of amplification tubes to be used and store the rest of them at 202C Two types of amplification tubes are shipped Mix 1 colorless tube for the amplification of the Coronavirus Metapneumoviru
21. s subtypes A and B Parainfluenza virus 1 2 3 and 4 subtypes A and B and RSV A WARNING The enzyme mixture should be added before the introduction of the extracted genetic material Mix 2 green tube for the amplification of Adenovirus Bocavirus Enterovirus Echovirus Influenza virus A B C y New FluA H1N1 2009 Metapneumovirus Rhinovirus y VRS B WARNING The enzyme mixture should be added before the introduction of the extracted genetic material e Enzyme mixture this is a mixture of the RT retrotranscriptase enzyme and DNA polymerase Ready to use Store at 209C Note The kit package includes a self adhesive and irreversible temperature indicator the appearance of a reddish color on the visualization window indicates that at a certain moment products have exceeded storage temperature of 20 C and they should not be used 3 3 Visualization reagents The visualization kit is shipped and should be stored at 42C WARNING Once received the CLART Strip CS should be stored at room temperature e CS strips including all specific probes They are provided in a sealed thermal envelope Store it closed at room temperature 25 C max protected from direct light e SH Hybridization Solution Store at 42C e DC Conjugate Diluent Store at 42C e CJ Conjugate Store at 42C Centrifuge once before use e RE Development Solution Store at 42C and protected from light e TL Wash Buffer Store at 42C
22. specificity Specificity experiments were performed with the 17 recombinant plasmids and it was observed that there were no cases of unspecific detection of viruses Therefore we consider a 100 analytical specificity Diagnostic utility parameters In order to determine the diagnostic parameters of the kit comparative evaluation was performed using the CLART PneumoVir and the most extended techniques used in hospitals Inmunofluorescense Inmunochromatography Q PCR Following hospitals collaborating with the evaluation 21 e Microbiology Department of the Germans Trias i Pujol Univertitary Hospital Badalona e Virology Unit of the Virgen de la Arrixaca Universitary Hospital e Virology Laboratory of the Reims Universitary Hospital France Genomic material was extracted from 296 nasopharyngeal lavages and analyzed for detecting the presence of every single virus in table 2 When both results alternative method and CLART PneumoVir showed same result result was considered as valid In case of discrepancies between both methods sequencing result was considered as valid In case of no sequencing availability discrepancies were analyzed with an in house Nested PCR followed up by sequencing Virus PneumoVir Sensitivity Specificity 100 00 100 00 88 24 100 100 00 100 00 100 00 100 00 100 00 100 00 100 00 100 00 86 67 100 100 00 100 00 98 15 99 55 83 33 100
23. ul diluted TL Solution and mix 10 to 15 times with the pipette Discard the diluted TL Solution using a pipette or a vacuum system leaving a volume In case the Thermoblock has not reached a temperature of 302C when you get to this step leave the tubes filled with diluted TL Solution until the Thermoblock reaches the necessary temperature 6 Blocking and adding conjugate It is recommended to centrifuge the CJ solution for 10 seconds before use Then prepare the diluted CJ solution To this end mix in a tube 1 mL of DC solution with 7 5 pl of CJ solution for each strip When stored at 42C the diluted CJ Solution remains stable within 4 hours after its preparation Do not use once this time has elapsed Add 100 ul of diluted CJ Solution to each well Incubate for exactly 15 minutes at 302C agitating at 550 rpm Following this incubation rapidly discard the solution out of the well using a pipette or vacuum system Lower the temperature of the Thermoblock to 252C for its use at step 9 15 7 Triple Washing Wash three times with 200 ul of diluted TL Solution to every well and mix it 10 to 15 times then discard the solution using a pipette or vacuum system If such washing is not performed rapidly it can cause an illegible signal during reading 8 Development with RE Solution Remove the TL solution add 100 ul of RE solution to each well of the CS and incubate for 10 minutes at 252C in the Thermoblock without agitating WARNING It is
24. very important to use the Thermoblock without agitating and read the samples immediately after incubation 9 Remove the RE Solution using a pipette or vacuum system The microarray should be dry 10 CAR CLINICAL ARRAY READER place the plate normally on the tray and the CAR will take and analyse the arrays automatically 7 4 2 autoclart visualization 1 Denaturation Place the amplification tubes in the thermocycler when this has reached 952C and incubate the tubes for 10 min Not to exceed 10 min time of denaturation to prevent the tubes are opened and contamination may occur Remove the tubes from the 952C incubation and place them immediately on ice 2 Switch on the autoclart unit and follow the instructions described on the screen 3 Closet the door and press the knob 4 Select Run at the main menu 5 Select the assay PneumoVir test among those listed 6 Select the well of the strip where run should start A1 or E1 in case the first 4 wells have been already processed 7 Select the amount of samples to be processed With autoclart user can process from 4 up to 96 samples per run in any case samples should be multiples of four 8 Confirm that number of samples and start up well A1 or E1 are correct 9 Place the tips rack full on its position 10 Check that both tip waste and liquid waste containers are empty 11 Fill the bottle with 250 ml distilled water 16 12 Add each reagent to its
25. with respiratory infections 7 2 Automatic extraction 7 3 Amplification reaction 7 4 Visualization of amplified product on CLART Strip CS 7 4 1 Manual visualization 7 4 2 autoclart visualization 8 RESULTS READING 9 RESULTS INTERPRETATION 10 TECHNICAL AND OPERATIONAL SPECIFICATIONS 11 BIBLIOGRAPHY 1 GLOSSARY 25 C C Check handling instructions Expiration date Medical Device for In Vitro Diagnostics Lot Store at room temperature Store between 42C to 82C Store between 302C to 182C 2 PROTOCOL DESCRIPTION CLART PneumoVir kit is capable of detecting and characterizing the presence of the 19 most frequent types of human viruses causing respiratory infections in the most common clinical samples including the specific detection of the Influenza subtype causing the new Influenza A H1N1 2009 Viruses analyzed include Adenovirus Bocavirus Coronavirus Enterovirus Echovirus Influenza virus A subtypes H3N2 human H1N1 human B C and H1N1 2009 Metapneumovirus subtypes A and B Parainfluenza virus 1 2 3 and 4 subtypes A y B Rhinovirus Respiratory Syncitial Virus type A VSR A Respiratory Syncitial Virus type B VSR B Virus detection is performed via RT PCR reverse transcriptase PCR amplification of a specific 120 330 bp fragment of the viral genome In order to avoid false negative results each PCR tube includes an Amplification Internal Control Its detection ensures the pr
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