User manual STIs-A
Contents
1. 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 used 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 contami2. 14 CAR CLINICAL ARRAY READER place the plate normally on the tray and the CAR will take and analyse the arrays 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 column lists the analytical result positive negative inconclusive without DNA or inhibited 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 19 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 amplificati
3. 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 well 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 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 Labelle d products Biotin SF SF tt 2 Conju gate Xo XP
4. 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 there 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 STIs A 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 Store samples at 4 C always being processed in a time less than 48h Otherwise stored frozen at 20 C 7 WORKING PROTOCOL 7 1 Extraction of genetic material 11 7 1 1 Manual extraction of genetic material Preparation procedures before starting the extraction e Buffer B3 preparation Transfer Buffer B1 into Buffer B2 and mix it thoroughly by pipetting up and down The resulting Buffer B3 is stable for 5 months when stored in the dark at room temperature e Dissolve lyophilised Proteinase K in BP before using in order to reach a stock concentration of 20 mg ml The
5. 8 100 Swabs Repeatability n 41 99 2 Reproducibility n 46 24 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 25
6. 9696 ethanol to each sample and vortex them immediately Note Do not discard any white precipitate that might form after adding the ethanol Along with the rest of the solution this precipitate should be added to the purifying column in the next step 6 Place a purifying column into a 2 ml collection tube for each sample Add the first tube of the samples into the purifying column Note In this step one of the tubes of the same sample processed in parallel since Step 1 to Step 5 are added to the purification column Centrifuge at 12 000 rpm for 1 min Make sure that all the solution has completely overpassed the membrane and discard the filtered solution and the 2 ml collection tube 7 Place the purifying column in a new 2 ml collection tube Add the second tube of the sample processed in parallel into the purifying column Note The two tubes of the same sample were processed in parallel since Step 1 to Step 5 In this step both are joined in the same purification column Centrifuge at 12 000 rpm for 1 min Make sure that all the solution has completely overpassed the membrane and discard the filtered solution and the 2 ml collection tube 13 8 Place the purifying column in a new 2 ml collection tube and add 500 yl of Solution BW to the column and centrifuge at 12 000 rpm for 1 min Dispose the filtered solution and the 2 ml collection tube 9 Place the purifying column in a new 2 ml collection tube and add 600 ul of Solution
7. Development reaction 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 The 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 Extraction and purification reagents e Extraction and Purification reagents are delivered to users at 42C or room temperature Components e Purification columns adapted to 2 ml tubes e 2mlCollection Tubes e Buffer T1 e Buffer B1 e Buffer B2 e Buffer BS e Buffer BW e Buffer BE e Label for Buffer B3 e Proteinase K lyophilized keep at 49C when resuspended e Buffer PB 3 2 Amplification reagents They are shipped and should be stored at 209C e Amplification tubes white tube Ready to use They contain 45 uL 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 Chlamy
8. 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 e Use the internal lysis and the Generic protocol following the instructions provided by the manufacturer of the NucliSENS 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 14 7 2 Amplification reaction Amplification specific recommendations e Work in the pre PCR area always using a cabinet and following the recommendations mentioned in section 5 1 e 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 C 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 micropipette Keep the tubes refrigerated at any time 4 Program the following temperature cycles on the thermocycler 1 cycle
9. 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 Sensitivity Specificity NPV Chlamydia trachomatis 99 6 30 Neisseria 226 1 1 98 99 6 gonorrhoea vu 6 270 99 6 genitalium Tv 53 223 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 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 Positive 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 23 Ln Repeatability n 6 100 Reproducibility n
10. 959C 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 909C This way possible unspecified amplifications due to incubation below hybridization 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 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 Make sure that before the hybridization begins the thermomixer temperature has reached the 569C for at least 1 hour 15 Warm up the SH hybridization solution in the thermomixer Prepare fresh wash solution before each assay do not reuse previously prepared solutions or residues Use filtered tips different for each well and change it every time a reagent is added 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 10 diluted bleach solution after every assay Make sure the pump aspirates properly and does not le
11. B5 to the column and centrifuge at 12 000 rpm for 1 min Dispose the filtered solution and the 2 ml collection tube 10 Place the purifying column in a new 2 ml collection tube and centrifuge once more at 12 000 rpm for 1 min in order to eliminate any remaining Solution B5 Note Any residual ethanol from Solution B5 might inhibit the required enzymatic reactions it must therefore be completely eliminated by centrifugation 11 Place the purifying column in a sterile 1 5 ml microcentrifuge tube Add 25 ul of Solution BE pre heated at 70 C and incubate for 3 minutes this solution in the column Note Be careful to add the 25 ul of Solution BE into the center of the membrane of the column Centrifuge for 1 minute at 12 000 rpm Recover the filtrate approximately 25 ul in the microcentrifuge tube Store the rest at 20 C Store the extracted DNA at 20 C 7 1 2 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 In all the cases transfer 1 ml of sample to the extractor e For each series of samples to be analyzed an extraction negative
12. CLART GEN MICA CLART STIs A DETECTION AND GENETIC IDENTIFICATION OF PATHOGENS CAUSING SEXUALLY TRANSMITTED INFECTIONS STIs FOR N VITRO DIAGNOSIS CLART STIs A CLART CLART Strip CAR SAICLART and AUTOCLART 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 Parque Empresarial Alvento Edificio B Calle V a de los Poblados 1 12 planta 28033 Madrid Spain WWW genomica com Version 5 July 2015 TABLE OF CONTENTS 3 1 3 2 3 2 3 3 4 1 4 2 5 1 5 2 5 2 6 7 GLOSSARY PROTOCOL DESCRIPTION KIT COMPONENTS AND STORAGE Extraction and purification reagents Amplification reagents Visualization reagents Other components MATERIALS REQUIRED BUT NOT PROVIDED Reagents and materials Equipment RECOMMENDATIONS AND HANDLING PROCEDURES General recommendations Precautions for the extraction and addition of extracted material to the amplification tube Precautions for visualization SAMPLES WORKING PROTOCOL 7 1 Extraction of genetic material 7 2 7 3 10 11 7 1 1 Manual extraction of genetic material 7 1 2 Automatic extraction of genetic material Amplification reaction Visualization of the amplified product 7 3 1 Manual visualization 7 3 2 autoclart visualiz
13. ation READING OF THE RESULTS INTERPRETATION OF THE RESULTS TECHNICAL AND OPERATIONAL SPECIFICATIONS BIBLIOGRAPHY 1 GLOSSARY Attention see instructions for use 2 Expiration date K In vitro diagnostic medical device H v EM Batch 25 C Store at room temperature 20 C e 1O C Store at 42C to 82C 4C m 18 C Store at 309C to 182C 30 C fee 2 PROTOCOL DESCRIPTION CLART STIs A 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
14. ave traces at the bottom of the well Aspirate the different solutions completely without touching the array 7 3 1 Manual visualization 1 Denaturation Place the amplification tubes in the thermocycler when this has reached 959C 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 959C incubation and place them immediately on ice 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 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 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 foa
15. dia trachomatis LGV Chlamydia trachomatis NLGV Chlamydia trachomatis generic Neisseria gonorrhoeae Mycoplasma genitalium and Trichomonas vaginalis It 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 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 3 Visualization reagents The visualization kit is shipped and should be stored at 49C 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 SH Hybridization Solution Store at 49C DC Conjugate Diluent Store at 49C CJ Conjugate Store at 49C Centrifuge once before use RE Development Solution Store at 49C and protected from light TL Wash Buffer Store at 49C Adaptor and lid for 8 well strips 3 4 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 CAR 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 di
16. e amplification tubes in the thermocycler when this has reached 959C and incubate the tubes for 10 min the tubes from the 959C incubation and place them immediately on ice 2 Switch on the autoclart unit and follow the instructions described on the screen 1 Closet the door and press the knob 2 Select Run at the main menu 3 Select the assay STIs Test among those listed 4 Select the well of the strip where run should start A1 or E1 in case the first 4 wells have been already processed 5 Select the amount of samples to be processed The autoclart allows to process from 4 up to 96 samples per run In any case samples should be always multiples of four 6 Confirm the number of samples and start up well A1 or E1 are correct 7 Place the tips rack full on its position 8 Check that both tip waste and liquid waste containers are empty 9 Fill the bottle with 250 ml distilled water 10 Add each reagent to its 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 befor
17. e 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 7 5 ul CJ reagent Vortex the diluted solution in order to mix it properly up 18 RE Developer Add the RE volume indicated on the display 11 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 12 For adding the samples on the CSs please remove the plate carefully from autoclart unit and add 5ul of the denatured product from the green tube and 5yl of the denatured product from the blue tube respectively to each well Mix it up carefully in order not to touch the array and place the microplate again on the autoclart Press the knob again to continue the visualization process 13 Once finished the visualization process the autoclart unit will beep indicating the end of the run Please remove the microplate carefully and proceed with the reading step on the CAR WARNING Once the automatic visualization phase is finished it is important to read the results immediately in the CAR otherwise false negatives may appear due to the loss of the sign
18. ed 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 CLART STIs A A negative result for the reference technique and a positive result for CLART STIs A the discrepancy 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 N 49 40 Chlamydia CTgeneric 1 trachomatis LGV 1 NLGV 38 Neisseria gonorrhoeae Mycoplasma genitalium Vu 0 49 o o 100 100 vaginalis CT Chlamydida trachomatis LGV Chlamydida trachomatis producers of lymphogranuloma venereum 22 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
19. ilities that may lead to an inconclusive result in which case there would be repeated from the visualization e In those cases when replicates of an array probe are very different from each other e 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 20 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 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 1 sensitivity Analytical Microorganism EE 2 Neisseria gonorrhoeae Chlamydia trachomatis 102 NLGV Mycoplasma genitalium Trichomonas vaginalis Chlamydia trachomatis LGV 10 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 pla
20. ming to each CS well Next add 5 uL of denatured PCR product from amplification tube Use one array per sample 16 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 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 Double Wash Add 200 pL 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 arrived at this step the thermomixer has not reached 30 C the wells are left with TL solution until the thermomixer reaches the temperature 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 stri
21. nation 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 order 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 10 5 2 Precautions for the extraction and addition of extracted material to the amplification tube 1 Always wear gloves Clean working surfaces of cabinets with a 1096 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 N 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
22. on 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 e Specific oligonucleotides for the targets of the pathogens 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 e Fault in the amplification tube in which case there would be repeated from the amplification There are two possib
23. p 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 C shaking at 550 rpm After this incubation 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 C and in motion so it may be used later in step 8 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 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 C in the thermomixer without shaking Warning It is very important to use the thermomixer without shaking 17 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 plate normally on the tray and the CAR will take and analyse the arrays automatically 7 3 2 autoclart visualization 1 Denaturation Place th
24. smids observing that an unspecific detection of other microorganisms different to what is sought to be determined is not produced Therefore it is considered that the technique reaches an analytical specificity of 100 Chlamydia trachomatis generic 21 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 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 e 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 consider
25. stributed by GENOMICA to be used exclusively with its diagnostic kits e SAICLART software developed and validated by GENOMICA for image processing e kit CLART STIs A specific software designed and validated by GENOMICA 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 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 e 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 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 e One adjustable micropipette ranging from 1 20 pL 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 Thermoblock with plate adapter lid and adjustable shaking at 25 C 30 C y 56 C Compatible with 96 well plates e Vortex e Vacuum system
26. tube with 1 ml of sample will be processed in parallel since Step 1 to Step 5 of the Manual Extraction Protocol after that the two tubes will be joined in the same purification column Urine sample 12 Agitate vigorously the sample container and take 2 ml of the sample Transfer 1ml of the sample into 1 5 ml microcentrifuge tube and the other 1 ml of the sample into the other 1 5 ml microcentrifuge tube Note The sample volume needed for manual extraction is 2 ml so each sample will have to be processed in parallel in two 1 5 ml microcentrifuge tubes Each tube with 1 ml of sample will be processed in parallel since Step 1 to Step 5 of the Manual Extraction Protocol after that the two tubes will be joined in the same purification column Manual extraction protocol 1 Centrifuge the samples for 10 min in a microcentrifuge at 12 000 rpm and remove all the liquid with a micropipette Be careful not to remove the precipitate 2 Resuspend the precipitate in 180 gl Solution T1 Transfer to a sterile 1 5 ml microcentrifuge tube 3 Add 25 ul of proteinase K solution and mix it all in a vortex Incubate the samples at 56 C for 1 hour in a thermomixer with agitation until the sample is completely lysed Vortexing samples every 15 minutes for a few seconds will accelerate lysis 4 After lysis add 200 ul of solution B3 to each sample Mix the samples thoroughly by vortexing them and incubate them at 70 C for 10 min 5 Add 210 ul of
27. volume of PB should be used is indicated on the bottle of proinase K Store it at 49C e Buffer B5 preparation Add 96 100 ethanol to the Buffer B5 bottle before use The volume of ethanol should be used is indicated on the bottle e Heat solution BE to 70 C before use e All centrifuging should be performed at room temperature unless otherwise stated Warning Solutions B3 and BW contain guanidine hydrochloride The use of gloves glasses and laboratory clothing is recommended when handling Sample preparation Swabs gt If the swab is conserved in dry medium add 2ml saline buffer 0 9 NaCl to the tube containing the swab and agitate vigorously with a vortex for 1 min Take 1 ml of the sample to a sterile 1 5 ml tube and other 1 ml of the same sample to the other sterile 1 5 ml tube Note The sample volume needed for manual extraction is 2 ml so each sample will have to be processed in parallel in two 1 5 ml microcentrifuge tubes Each tube with 1 ml of sample will be processed in parallel since Step 1 to Step 5 of the Manual Extraction Protocol after that the two tubes will be joined in the same purification column f the swab is conserved in liquid medium take 2 ml of the sample Take 1ml into a sterile 1 5 ml tube and the other 1ml into another sterile 1 5 ml tube Note The sample volume needed for manual extraction is 2 ml so each sample will have to be processed in parallel in two 1 5 ml microcentrifuge tubes Each
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