CLART STIs A v2 English
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1. EasyMag DNA of Biomerieux automatic extractor The elution volume must be 25 ul If another extraction system is used the elution volume should be optimized in the range of 20 30 ul 7 2 Amplification reaction Amplification specific recommendations Work in the pre PCR area always using a cabinet and following the recommendations mentioned in section 5 1 DNA always adds in cabinet and following the recommendations mentioned in section 5 1 During the process keep tubes separate and refrigerated 1 Thaw an amplification tube white for each sample to analyze Keep on ice and not use temperatures above 37 for thawing 2 Centrifuge the amplification tubes for a few seconds so that all liquid can get to the bottom of the tubes in case you don t have microcentrifuge adaptors available for the tubes you can use larger tubes after having cut their cap off 3 Add 5 uL of the extracted DNA to every amplification tube and mix several times with the 11 micropipette Keep the tubes refrigerated at any time 4 Program the following temperature cycles on the thermocycler 1 cycle 95 15 min 959C 30 sec 45 cycles 639C 60 sec 729C 60 sec 1 cycle 729C 10 min 49C continuously until tube collection optional 5 Start the program and place the tubes in the thermocycler when the block has exceeded 90 This way possible unspecified amplifications due to incubation below hybridization2. plate with the plastic lid provided and incubate in the thermomixer for 1 hour at 56 C shaking at 550 rpm After this incubation remove the plate from thermoshaker and aspirate the SH solution of the CS with a pipette or preferably with a vacuum pump The array must be free from solution residues although it must never remain dry Add the next solution immediately After incubation set the thermomixer at 30 C and in motion so it may be used later in step 6 5 Double Wash Add 200 of diluted TL solution to each CS well resuspend 10 to 15 times with the multichannel pipette Discard the diluted TL solution with a pipette or preferably with a multichannel vacuum pump without leaving any residues Repeat the procedure This step must be carried out with different tips for each well in both washes If having 13 arrived at this step the thermomixer has not reached 30 C the wells left with TL solution until the thermomixer reaches the temperature 6 Blocking and conjugate It is recommended to centrifuge the high affinity CJ solution for 10 seconds before use Then prepare the diluted CJ solution as follows for each CS strip mix 1 mL of DC solution and 7 5 pL of high affinity CJ solution Discard the diluted TL Solution without leaving any residues of the solution and add 100 uL of diluted CJ solution to each CS well Incubate for 15 exact minutes in the thermoshaker at 30 shaking at 550 rpm After this incubati
3. to prevent the tubes are opened and contamination may occur Remove the tubes from the 959C incubation and place them immediately on ice 2 Diluted TL solution preparation For each CS strip a total of 8 wells prepare 10 mL of diluted wash solution by adding 1 mL of TL solution to 9 mL of distilled water gently stir 3 Prewash of the CS Before beginning the assay it is necessary to wash the strips by adding 200 uL of diluted TL solution to each well Mix it with the multichannel pipette 10 to 15 times taking into account that the surface of the array must not be touched It is advised to carry out this wash while the amplified samples are being denatured and maintain the wash solution in the well until samples are going to be added Discard the diluted TL solution with a pipette or preferably with a vacuum pump The array must be free from solution residues although it must never remain dry Add the next solution immediately 4 Hybridization Before using the SH solution it must be heated at 56 C until the complete dilution of the salts Once the PCR products have been denatured add 100 uL of SH solution prevent foaming to each CS well Next add 5 uL of denatured PCR product from amplification tube Use one array per sample Mix it several times being careful not to touch the bottom of the well It is recommended to load each strip independently and separately from the rest to avoid contaminations Cover the microtiter
4. 7 Sensitivity Specificity PPV NPV vp FP FN 42 8 16 4 234 0 1 97 7 100 100 996 NLGV 30 MS 49 226 1 1 98 99 6 98 99 6 gonorrhoea 6 270 0 1 85 7 100 100 99 6 genitalium 53 223 0 1 98 1 100 100 99 6 vaginalis CT Chlamydida trachomatis LGV Chlamydida trachomatis producers of lymphogranuloma venereum NLGV Chlamydida trachomatis no producers of lymphogranuloma venereum VP True positive VN True negative FP False positive 18 FN False negative The number of samples analysed does not match the number of micro organisms analyzed since in these 277 samples are available there are some negative mono infeccion samples and samples with co infection of 2 or more microorganisms Unable to confirm the presence of the organism in the sample with Nested PCR and or specific PCR Dositive sample for sequencing Neisseria flava Diagnostic specificity The technique has been validated with urine and swabs samples both negative and positive for other microorganisms not included in the kit and the results show no cross reaction with them Reproducibility and diagnostic repeatability by sample type The reproducibility and diagnostic repeatability was processed from sample extraction to visualization in CS Urines Repeatability n 6 100 Reproducibility n 8 100 Swabs Repeatability n 41 99 2 Repr
5. CLART STIs A DETECTION AND GENETIC IDENTIFICATION OF PATHOGENS CAUSING SEXUALLY TRANSMITTED INFECTIONS STIs FOR JN VITRO DIAGNOSIS CLART STIs Amplification 48 tests Ref CS 1112 48 16 tests Ref CS 1112 16 Detection 48 test Ref CS 1212 48 16 test Ref CS 1212 16 CLART and CLART Strip are registered Trademarks of GENOMICA For more information please refer to the web site www genomica com Only Chlamydia trachomatis diagnostic has been assented by the NB 0318 CE for other microorganisms GENOMICA S A U Alcarria 7 28823 Coslada Madrid Spain Tel 34 91 674 89 90 Fax 34 91 674 89 91 Version 2 August 2013 TABLE OF CONTENTS 1 GLOSSARY 2 PROTOCOL DESCRIPTION 3 KIT COMPONENTS AND STORAGE 3 1 Amplification reagents 3 2 Visualization reagents 3 3 Other components 4 MATERIALS REQUIRED BUT 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 2 Precautions for visualization 6 SAMPLES 7 WORKING PROTOCOL 7 1 Automatic extraction of genetic material 7 2 Amplification reaction 7 3 Visualization of the amplified product 8 READING OF THE RESULTS 9 INTERPRETATION OF THE RESULTS 10 TECHNICAL AND OPERATIONAL SPECIFICATIONS 11 BIBLIOGRAPHY 1 GLOSSARY Attention see instructions for use Expiratio
6. O Precipitation of substrate MBO Figure 2 Diagram of the visualization method Probes immobilized on the surface capture their complementary biotin labelled amplified products With the help of biotin they bind to the conjugate in this case streptavidine HRP HorseRadish Peroxidase The o dianisidine substrate by the action of the HRP produces a precipitate on the area where hybridization occurs 3 KIT COMPONENTS AND STORAGE CLART STIs A kit contains reagents enough for performing 16 or 48 clinical samples analysis The reagents included in the kit have been grouped into various packages depending on the temperature at which they should be stored When storage recommendations are observed all reagents should remain stable until kit s expiration date 3 1 Amplification reagents They are shipped and should be stored at 209C e Amplification tubes white tube Ready to use They contain 45 pL of reaction mixture Thaw on ice the exact number of amplification tubes that will be used and keep the rest at 209C This tube allows the amplification of Chlamydia trachomatis LGV Chlamydia trachomatis NLGV Chlamydia trachomatis generic Neisseria gonorrhoeae Mycoplasma genitalium and Trichomonas vaginalis t also includes the amplification of an amplification control and an extraction control The kit package includes a self adhesive and irreversible temperature indicator the appearance of a reddish colour on
7. croorganisms different to what is sought to be determined is not produced Therefore it is considered that the technique reaches an analytical specificity of 100 Diagnostic utility parameters In order to determine the diagnostic parameters of the kit a comparative assessment of the CLART STIs A kit was carried out against the reference techniques culture or PCR For this evaluation we collaborated with the following laboratories Department of Microbiology Trias i Pujol University Hospital Badalona Spain e OpenHouse Medical Centre Madrid Spain e Monte Naranco Hospital Oviedo Spain e Sandoval Sanitary Center Madrid Spain e Virgen de la Macarena Hospital Sevilla Spain The presence of each one of the microorganisms that were detected with the kit was analyzed from genetic material of urine and swab samples We analyzed a total of 326 samples 49 urine samples and 277 swabs samples The organisms tested are described in Table 2 For each sample the result was considered true if there is concordance between the reference technique and the CLART STIs A technique The discrepancies between the two techniques were resolved as follows A positive result for the gold standard and negative result for CLART STIs A the reference technique is considered like correct data and it is considered false negative for STIs CLART A A negative result for the reference technique and a positive result for CLART STIs A the di
8. d outside these areas 1 Pre PCR area DNA extraction sample preparation and addition of the extracted material to the amplification tubes are performed in this area Sample manipulation must be carried out within a biosafety cabinet BSC 2 Post PCR area Amplification and visualization of the amplified product are carried out in this area The material of this area should never come into contact with the material of the extraction area Avoid entering the pre PCR area after having worked in the visualization area 2 Always use gloves It is recommended to change gloves quite frequently and it is mandatory to change gloves before start working in each of the aforementioned areas New gloves must always be used when DNA is added to the amplification tubes 3 Clean working areas laboratory cabinets hoods grids pipettes thoroughly with a 1096 diluted bleach solution after every sample batch processing it is mandatory to disinfect all working areas in case of contamination For thermocyclers and thermomixers it is advised to clean them before and after used in these same conditions 4 Always use filter tips and positive displacement pipettes to avoid contamination due to micropipettes Different sets of pipettes should be used in each area 5 Use disposable and autoclaved laboratory material 6 Never mix reagents from two different vials even if they belong to the same lot 7 Close reagent tubes immediately after use in orde
9. ens to be detected 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 the DNA extraction from stool sample was conducted successfully even if there was no amplification of pathogen PCR control would only be essential when no amplification in the tube is found 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 Presence of inhibitors in the sample in which case there would be repeated from extraction Fault in the amplification tube in which case there would be repeated from the amplification There are two possibilities that may lead to an inconclusive result in which case there would be repeated from the visualization 15 In those cases when replicates of an array probe very different from each other 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 10 TECHNICAL AND OPERATIONAL SPECIFICATIONS 10 1 Control of known inte
10. er 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 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 Saline solution 0 9 NaCl 4 2 Equipment Microcentrifuge Thermocycler Laminar flow chamber for the extraction laboratory Three adjustable micropipettes ranging from 1 20 uL 20 200 uL and 200 1000 uL for the extraction laboratory One adjustable micropipette ranging from 1 20 to add the genetic material to the amplification tubes Three adjustable micropipettes ranging from 1 20 pL 20 200 and 200 1000 for the visualization laboratory Thermoblock with plate adapter lid and adjustable shaking at 25 30 y 56 C Compatible with 96 well plates Vortex Vacuum system 5 RECOMMENDATIONS AND HANDLING PROCEDURES Read carefully before starting the assay in order to avoid contamination Read carefully before beginning the technique 5 1 General recommendations 1 This assay should be performed in two 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 use
11. here are no bubbles fibers or spots interfering with the reading Otherwise clean the outer face of the well with cellulose paper 6 SAMPLES The CLART 5715 kit has been designed and validated to be used with DNA extracted from urine and swabs anal vaginal endo cervical urethral and faringeal GENOMICA is not responsible for the results obtained if other types of samples are used 10 Store samples at 4 C always being processed time less than 48h Otherwise stored frozen at 20 C 7 WORKING PROTOCOL 7 1 Automatic extraction in the NucliSENS EasyMag DNA of Biomerieux e For swab samples if the preservation medium of the swabs is liquid take 1 ml of the sample for DNA extraction If it is stored in dry medium add 1 5 ml of saline solution 0 996 sodium chloride to the tube containing the swab and vortex vigorously for 1 minute Load 1 ml for DNA extraction e For urine samples shake the container and load 1ml of the sample for DNA extraction e cases transfer 1 ml of sample to the extractor For each series of samples to be analyzed an extraction negative control 0 996 sodium chloride must be included to verify that the samples have not been contaminated during the extraction amplification and visualization processes which might lead to a false positive result Use the internal lysis and the Generic protocol following the instructions provided by the manufacturer of the NucliSENS
12. ists in 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 The CLART STIs A detection system is based on the precipitation of an insoluble product in those microarray areas in which hybridization of amplification products with specific probes takes place During PCR amplified products are labelled with biotin After amplification these products are hybridized with their respective specific complementary probes that are immobilised in specific and well known microarray areas Afterwards they are then incubated with a streptavidine peroxidase conjugate The conjugate is bound through streptavidine with the biotin present in the amplified products which are bound to their specific probes and the peroxidase activity prompts the appearance of a non soluble product in the presence of the o dianisidine substrate which precipitates on the microarray areas where hybridization occurs Figure 2 Probes on the array Labelled product t E ae Biotin 1524 Y Y Y 4 n S PY F gt wr ai x Yer PP Hybridization My tr 3 E E 33 ff 7 Incubation 5 i Conjugate with conjugate f Development reaction n O0 5 22280 E 7 7 LI S3 D H s
13. mn lists the analytical result positive negative inconclusive without DNA or inhibited 14 9 INTERPRETATION OF THE RESULTS One of the main drawbacks of genomic amplification is the utilization of poor quality DNA samples for taking insufficient amount of sample DNA degradation due to an incorrect storage of the sample loss of DNA of the sample during extraction or the presence of DNA polymerase inhibitors in the samples to be analyzed thus interfering with the genomic amplification and resulting in false negatives With the CLART STIs A kit the false negatives are removed by adding an internal control in the amplification tube which is indicative of the efficiency of the amplification reaction Also it is included in the tube a genomic DNA extraction control to detect false negatives due to failures in the extraction 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 The amplification tube contains the following amplification primers A pair of oligonucleotides that amplify a fragment of the human CFTR gene as a genomic DNA control of the patient A pair of oligonucleotides that amplify a modified plasmid included in the amplification tube which is used as amplification control of the PCR reaction Specific oligonucleotides for the targets of the pathog
14. n date In vitro diagnostic medical device IVD NUN Batch Store at room temperature 20 8 Store at 49C to 89C 4 C Store at 309C to 182C 30 2 PROTOCOL DESCRIPTION CLART STIs detects the presence microorganisms causing sexually transmitted infections in clinical samples urine and swabs The microorganisms detected are detailed in the list below e Chlamydia trachomatis It will distinguish between the two serotypes groups o Producers of lymphogranuloma venereum LGV include serotypes L1 L2 and L3 o Non producers of lymphogranuloma venereum NLGV include serotypes D K It will include a generic detection of Chlamydia trachomatis for those cases in which the serotype is not achieve e Neisseria gonorrhoeae e Mycoplasma genitalium e Trichomonas vaginalis Detection is carried out by the specific amplification of each microorganism in the sample originating a variable fragment between 100 and 300 base pairs To avoid false negatives the tube contains an amplification internal control which ensure the correct working of the tube and a genomic DNA extraction control from the sample which guarantee that genetic material has been isolated during the extraction process The detection of the product amplified by PCR is carried out by means of a low density microarray platform CLART Clinical Array Technology The platform is based a very simple principle but at the same time cost effective It cons
15. oducibility n 46 97 1 19 11 BIBLIOGRAPHY 1 Gardnerella Trichomonas vaginalis Candida Chlamydia trachomatis Mycoplasma hominis and Ureaplasma urealyticum in the genital discharge of symptomatic fertile and asymptomatic infertile women Erminia Casari Antonella Ferrario Emanuela Morenghi Alessandro Montanelli New Microbiologica 33 69 76 2010 2 Global strategy for the prevention and control of sexually transmitted infections 2006 2015 breaking the chain of transmission WHO 3 Sexually Transmitted Diseases in the United States 2008 National Surveillance Data for Chlamydia Gonorrhea and Syphilis CDC 4 Persistent increase in the incidence of acute male urethritis diagnosed in general practices in France V ronique Massari Yves Dorl ans and Antoine Flahault British Journal of General Practice 2006 56 110 114 5 Mycoplasma genitalium presence resistance and epidemiology in Greenland Dionne C Gesink Gert Mulvad Ruth Montgomery Andersen Upaluk Poppel Stephan Montgomery Andersen Aka Binzer Lee Vernich Gillian Frosst Flemming Stenz Elizabeth Rink Ove Rosing Olsen Anders Koch and J rgen Skov Jensen Int J Circumpolar Health 2012 71 18203 20
16. on remove the plate and discard the solution rapidly with a pipette or a multichannel vacuum pump Once finished the incubation set the thermomixer at 25 and in motion so it may be used later in step 8 7 Triple Wash Add immediately 200 uL of diluted TL solution to each CS well mixing it 10 to 15 times with the multichannel pipette and discard the solution completely with the pipette or vacuum pump Repeat the procedure two more times It is very important to avoid any residues of the CJ solution since they could react with the RE Solution generating an unspecified signal 8 Development with RE solution Remove the diluted TL solution completely and add 100 uL of RE solution to each CS well and incubate for 10 minutes at 25 in the thermomixer without shaking Warning It is very important to use the thermomixer without shaking 9 Discard the complete TL solution using a pipette or a vacuum system The array must remain dry for the reading 10 CAR Clinical Array Reader Place the adaptor in the CAR tray and the plate on top Once the tray is closed the reading is performed automatically 8 READING OF THE RESULTS The processing of data obtained from each analysis is carried out automatically The reading and analysis system CAR will provide a report indicating the results The monitor displays a table with two columns in the left column lists the species that are characterized in the array In the right colu
17. r to avoid contamination 8 Discard the micropipette tip after pipetting 9 GENOMICA is not responsible for the results obtained with the kit if other samples different to the ones indicated are used 5 2 Precautions for the extraction and addition of extracted material to the amplification tube Always wear gloves e Clean working surfaces of cabinets with a 10 diluted bleach solution e 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 e The preparation of the samples before extraction must be made inside the cabinet 5 3 Precautions for visualization 1 Avoid the pipette tip or the vacuum system touching the bottom of the well since this could damage the probes printed at the well s bottom 2 It is recommended to add all solutions to the wall of the CS well never directly at the bottom 3 It is convenient not to add the SH solution hybridization solution until the denatured products of PCR are ready 4 The array must not remain dry 5 Following incubation with the CJ solution it is very important to wash the microarray thoroughly in order to avoid any residues that could react with the RE solution resulting in a non specific precipitation that could lead to false interpretations of the result 6 Avoid foaming when adding any reagent 7 When visualizing the image in the reader ensure that position markers appear and that t
18. rferences False negatives are one of the drawbacks in the detection by genomic amplification due to either 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 Analytical sensitivity Analytical sensitivity has been determined by the amplification of serial dilutions of recombinant plasmids for each one of the mutations detected by the kit Each one of them has the amplified product inserted including the part that is complementary to the specific detection probes The visualisation was done in CS giving rise to the following results Table 1 Microorganism Neisseria gonorrhoeae Chlamydia trachomatis 10 NLGV Mycoplasma genitalium Trichomonas vaginalis Chlamydia trachomatis LGV 10 Chlamydia trachomatis generic 16 Table 1 Relationship of the number of copies of recombinant plasmid necessary to obtain a sensitivity of 100 in the detection of each one of the microorganisms Analytical specificity Specificity experiments were carried out in recombinant plasmids observing that an unspecific detection of other mi
19. screpancy will be discussed by Nested specific PCR and sequencing The result is what is considered as true Table 2 Diagnostic sensitivity and specificity of CLART STIs A for each microorganism in urine samples Sensitivity Specificity PPV NPV N 49 VP VN FP FN 6 09 po uu T 9 0 0 100 100 100 100 trachomatis CTgeneric 1 17 LGV 1 NLGV 38 Neisseria 8 41 010 100 100 100 100 gonorrhoeae Mycoplasma 8 40 110 100 88 9 100 97 6 genitalium Trichomonas 0 491010 100 100 vaginalis CT Chlamydida trachomatis LGV Chlamydida trachomatis producers of lymphogranuloma venereum NLGV Chlamydida trachomatis no producers of lymphogranuloma venereum VP True positive VN True negative FP False positive FN False negative The number of samples analysed does not match the number of micro organisms analyzed since in these 49 samples are available there are some negative mono infeccion samples and samples with co infection of 2 or more microorganisms Trichomonas vaginalis in urine is not present so it has not been possible to analyze samples positive for this mo The detection is specific for the microorganisms given that not to appear false positive Table 3 Diagnostic sensitivity and specificity of CLART STIs A for each microorganism in swabs samples N 27
20. temperature are minimized 7 3 Visualization of the product amplified in CLART Strip CS Specific recommendations before starting visualization THE PROTOCOL DESCRIBED BELOW SHOULD ALWAYS BE PERFORMED IN THE POST PCR AREA DO NOT TAKE THE AMPLIFIED PRODUCT IN THE PRE PCR AREA 1 Turn on the CAR Clinical Array Reader before starting the whole procedure The self calibration of the equipment takes a few minutes and it is also necessary to introduce the name of the samples in the program before the reading 2 Make sure that before the hybridization begins the thermomixer temperature has reached the 569C for at least 1 hour 3 Warm up the SH hybridization solution in the thermomixer 4 Prepare fresh wash solution before each assay do not reuse previously prepared solutions or residues 5 Use filtered tips different for each well and change it every time a reagent is added 6 In case of using vacuum pumps equipped with 8 tip comb for aspirating solutions discard the combs after each use or decontaminate them with a 1096 diluted bleach solution after every assay Make sure the pump aspirates properly and does not leave traces at the bottom of the well 7 Aspirate the different solutions completely without touching the array VISUALIZATION 12 1 Denaturation Place the amplification tubes in the thermocycler when this has reached 95 and incubate the tubes for 10 min Not to exceed 10 min time of denaturation
21. the visualization window indicates that at a certain moment products have exceeded the storage temperature of 20 C and they should not be used 3 2 Visualization reagents The visualization kit is shipped and should be stored at 4 WARNING Once received the strips 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 max protected from direct light e 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 e Adaptor and lid for 8 well strips 3 3 Other components For the capture and subsequent processing of the image a reader unit running a tailor made software and the plate adaptor are required e Clinical Array Reader it allows for the automatic reading and interpretation of up to 12 CS i e up to a maximum of 96 samples It is manufactured and distributed by GENOMICA to be used exclusively with its diagnostic kits e An adaptor support that should be placed on the CAR tray before the scanning e SAICLART software developed and validated by GENOMICA for image processing e CLART STIs specific software designed validated by GENOMICA CAR Clinical Array Read
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