MTB Real TM RG SC IQ MX A LC Eng NEW ver - bio
Contents
1. Validity Interpretation lt 38 38 gt 38 Valid M tuberculosis complex is detected lt 38 Valid M tuberculosis complex is not detected gt 38 gt 38 Invalid Invalid repeat material sampling gt 38 lt 38 Invalid Equivocal repeat material sampling e result is positive in FAM Green channel lt 38 and the result is positive Ctx38 or negative Ct gt 38 in the JOE Yellow HEX Cy3 channel the result is valid Mycobacterium tuberculosis DNA is detected e the result is negative in the FAM FAM 490 channel and the result is positive in the JOE JOE 530 channel Ctx38 the result is valid Mycobacterium tuberculosis DNA is not detected e f the result is negative or Ct gt 38 in both JOE JOE 530 and FAM FAM 490 channels the result is invalid It is necessary to repeat amplification If the result is the same repeat DNA extraction If the result is the same again it is considered to be invalid In this case it is recommended to repeat material sampling e fthe result is Ct gt 38 in the FAM FAM 490 channel and the result is positive 1 lt 38 in the JOE JOE 530 channel the result is invalid It is necessary to repeat amplification If the result is the same repeat DNA extraction If the result is the same again it is considered to be equivocal In this case it is recommended to repeat material sampling Example of results FAM Green channel the samples contain MTB DNA JOE Yellow cha2. DNA extraction procedure 2 Weak Ct 35 or no signal of the Positive Control e The PCR conditions didn t comply with the instructions Check the amplification protocol and select the fluorescence channel reported in the manual 3 Fam Green signal with Negative Control of extraction Contamination during DNA extraction procedure All samples results are invalid Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol Use only filter tips during the extraction procedure Change tips between tubes Repeat the DNA extraction with the new set of reagents 4 Any signal with Negative Control of PCR DNA buffer Contamination during PCR preparation procedure All samples results are invalid Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol or special DNA decontamination reagents Pipette the Positive control at last Repeat the PCR preparation with the new set of reagents Sacace Real TM VER 21 03 2013 Sacace Real TM VER 21 03 2013 KEY TO SYMBOLS USED List Number LOT Lot Number For in Vitro Diagnostic Use Store at Manufacturer Consult instructions for use Expiration Date 1 8 5 SaCycler is a registered trademark of 580806 Biotechnologies and iQ5 are registered trademarks of Bio Rad Laboratories Rotor Gene is a registered trademark of Qiagen MX3005P is a reg
3. of 5 days or frozen at 20 80 C Sacace Real TM VER 21 03 2013 PROTOCOL 1 Prepare required quantity of reaction tubes or PCR plate for samples and controls 2 Prepare in the new sterile tube for each sample 10 N 1 pl of PCR mix 1 5 N 1 pl of PCR Buffer Flu 0 5 N 1 pl of TaqF DNA Polymerase and 0 5 N 1 pl of UDG Enzyme Vortex and centrifuge briefly 3 Add to each tube 15 pl of Reaction Mix 4 Add 10 pl of extracted DNA to appropriate tube Prepare for each panel 2 controls e 10 pl of DNA buffer to the tube labeled Amplification Negative Control e add 10 pl of C 8 IC to the tube labeled Amplification Positive Control 6 Insert the tubes in the thermalcycler Amplification 1 Create a temperature profile on your instrument as follows Stage Temp Time Fluorescence detection Hold 95 15 min 1 95 15s Cycling 65 30s 5 72 15s 95 15s Cycling 2 65 30 s 40 72 15 For example SaCycler 96 Sacace Rotor Gene 3000 6000 Q Corbett Research Qiagen CFX iQ5 BioRad Mx3005P Agilent ABI 7300 7500 StepOne Real Time PCR Applied Biosystems SmartCycler amp Cepheid LineGenek Bioer Fluorescence is detected at the 2nd step of Cycling 2 stage 60 C in FAM Green and JOE Yellow Hex Cy3 fluorescence channels Sacace MTB Real TM VER 21 03 2013 INSTRUMENT SETTINGS Rotor ty
4. 6 BIOTECHNOLOGIES u REF REF For in Vitro Diagnostic Use MIB Real TM HANDBOOK Heal Time PCR kit for the detection of Mycobacterium tuberculosis complex B15 50FRT TB15 50FRT so Sacace Real TM VER 21 03 2013 Sacace Real TM 21 03 2013 NAME MTB Real TM INTRODUCTION Tuberculosis abbreviated as TB for tubercle bacillus is a common and deadly infectious disease caused by mycobacteria mainly Mycobacterium tuberculosis Tuberculosis most commonly attacks the lungs as pulmonary TB but can also affect the central nervous system the lymphatic system the circulatory system the genitourinary system bones joints and even the skin Other mycobacteria such as Mycobacterium bovis Mycobacterium africanum and Mycobacterium microti can also cause tuberculosis Over one third of the world s population has been infected by the TB bacterium and new infections occur at a rate of one per second Not everyone infected develops the full blown disease asymptomatic latent TB infection is most common However one in ten latent infections will progress to active TB disease which if left untreated kills more than half of its victims In 2004 mortality and morbidity statistics included 14 6 million chronic active TB cases 8 9 million new cases and 1 6 million deaths mostly in developing countries In addition a rising number of people in the developed world are contracting
5. a new 1 5 ml tube e whole blood collected in either ACD or EDTA tubes e liquor stored in Eppendorf tube e liquid stored in Eppendorf tube e urine sediment use the intermedium part of stream e pleuric versament stored in Eppendorf tube e mycobacterium liquid culture conserved in Trilon B Specimens can be stored at 2 8 C for no longer than 48 hours or freeze at 20 C to 80 Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents DNA ISOLATION The following kit is recommended gt DNA RNA Prep Sacace REF K 2 9 Please carry out DNA extraction according to the manufacture s instruction Add 10 ul of Internal Control during DNA isolation procedure directly to the sample lysis mixture Sacace Real TM VER 21 03 2013 SPECIMEN AND REAGENT PREPARATION 1 Prepare required number of 1 5 ml disposable polypropylene micro centrifuge tubes including one tube for Negative Control of Extraction Negative Control C N B if the sample is sputum use the same 1 5 ml sample tube obtained from sputum preparation procedure see SAMPLE COLLECTION STORAGE AND TRANSPORT 2 Add to each tube 10 pl of MTB IC Internal Control 300 pl of Lysis Sol 3 Add 100 ul of samples to the appropriate tubes using pipette tips with aerosol barriers Prepare Controls as follows o add 100 pl of Nega
6. e avoided as this may reduce the sensitivity Components stored under conditions other than those stated on the labels may not perform properly and may adversely affect the assay results QUALITY CONTROL In accordance with Sacace s ISO 13485 Certified Quality Management System each lot is tested against predetermined specifications to ensure consistent product quality Sacace MTB Real TM VER 21 03 2013 WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device For n Vitro Diagnostic Use Only The user should always pay attention to the following s Lysis Solution contains guanidine thiocyanate Guanidine thiocyanate is harmful if inhaled or comes into contact with skin or if swallowed Contact with acid releases toxic gas Xn R 20 21 22 36 37 38 S 36 37 39 Component Prec Sol contains 2 propanol flammable Irritant R10 36 67 S7 16 24 25 26 Avoid contact with skin and eyes S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice Use sterile pipette tips with aerosol barriers and use new tip for every procedure e Store extracted positive material samples controls and amplicons away from all other reagents and add it to the reaction mix in a separate area e Thaw all components thoroughly at room temperature before starting an assay e When thawed mix the components and centrifuge briefly Use disposable gloves laboratory coats and eye protection when hand
7. ion system which increase the detection rate by 1796 Culture have high sensitivity and specificity however due to a slow growing tendency it takes 2 12 weeks to get a result The indirect methods such as X ray diagnostics CAT tuberculin diagnostics detection of tuberculosis antibodies do not directly identify TB however they give an understanding of current changes in organs Sacace Real TM VER 21 03 2013 INTENDED USE kit MTB Real TM is a test for Real Time qualitative detection of Mycobacterium tuberculosis complex M tuberculosis M africanum M bovis M bovis BCG M microti in the sputum urine bronchial lavages tissue and other biological materials PRINCIPLE OF ASSAY kt MTB Real TM is a Real Time Amplification test for the qualitative detection of Mycobacterium tuberculosis complex in biological materials Mycobacterium tuberculosis DNA is extracted from samples amplified using Real Time Amplification and detected using fluorescent reporter dye probes specific for M tuberculosis and M tuberculosis IC M tuberculosis IC is DNA fragment of IS 6110 insertion of Mycobacterium tuberculosis modified and cloned in bacteriophage A containing DNA fragments used in the kit as matrix for primers Internal Control IC serves as an amplification control for each individually processed specimen and to identify possible reaction inhibition IC is detected in a channel other than the M tuberculosis DNA MTB Real TM kit con
8. istered trademark of Agilent Technologies ABI is a registered trademark of Applied Biosystems LineGenek is a registered trademark of Bioer SmartCycler is a registered trademark of Cepheid Sacace Biotechnologies Srl VER NCA NCE C Caution Contains sufficient for lt n gt tests Version Negative Control of Amplification Negative control of Extraction Positive Control of Amplification Internal Control via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com Sacace Real TM VER 21 03 2013
9. ling specimens and reagents Thoroughly wash hands afterwards Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas Do not use a kit after its expiration date Dispose of all specimens and unused reagents in accordance with local authorities regulations e Specimens should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices Clean and disinfect all sample or reagent spills using a disinfectant such as 0 5 sodium hypochlorite or other suitable disinfectant Avoid sample or reagent contact with the skin eyes and mucous membranes If skin eyes or mucous membranes come into contact rinse immediately with water and seek medical advice immediately Material Safety Data Sheets MSDS are available on request Use of this product should be limited to personnel trained in the techniques of DNA amplification e The laboratory process must be one directional it should begin in the Extraction Area and then move to the Amplification and Detection Areas Do not return samples equipment and reagents to the area in which the previous step was performed Some components of this kit contain sodium azide as a preservative Do not use N metal tubing for reagent transfer Only for Module No 2 Sacace MTB Real TM VER 21 03 2013 PRODUCT USE LIMITATIONS All reagents may exclusively be used in in vitro diagnostics U
10. nnel the samples contain Internal Control B Oma Tute A sepe Cone Fit yd erm tetro pd Seve Oetats E 2 12 3 3 Jta Adu 4D abe Auto Scale Del aut Scale Log Scale Sacace MTB Real TM VER 21 03 2013 ANALYTICAL CHARACTERISTICS ANALYTICAL SPECIFICITY Analytical specificity of the primers and probes was validated with 110 Mycobacterium tuberculosis complex negative samples They did not generate any signal with the specific TB primers and probes The potential cross reactivity of the kit MTB Real TM was tested against the group control listed in the following table It was not observed any cross reactivities with these pathogens Control group Results Results Fam channel Joe Hex Cy3 channel Mycobacterium intracellulare Mycobacterium flei Mycobacterium scrofulaceum Mycobacterium kansasii z 7 Mycobacterium paratuberculosis Mycobacterium fortuitum Mycobacterium avium Escherichia coli z Staphylococcus aureus Streptococcus sp Clostridium diphtheriae Brucella sp Chlamydia trachomatis Chlamydia pneumonie The analytical specificity was also validated by DNA amplification of control strains of Mycobacterium tuberculosis complex Mycobacterium tuberculosis strains 192 5281 1443 328 330 932 350 1579 1528 1532 352 1030 Mycobacterium bo
11. pe instruments Calibrate Gain More Settings Optimisation Outlier Removal siope Correct FAM Green from 5 E to 10 0 03 10 JOE Yellow from i to 8 0 05 15 Off Plate type instruments The threshold line should cross only sigmoid curves of signal accumulation of positive samples and should not cross the baseline otherwise the threshold level should be raised Set the threshold at a level where fluorescence curves are linear and do not cross curves of the negative samples DATA ANALYSIS The fluorescent signal intensity is detected in two channels Mycobacterium tuberculosis is detected on the FAM Green channel C DNA on the JOE Yellow HEX Cy3 channel Interpretation of results The results are interpreted through the presence of crossing of fluorescence curve with the threshold line Analysis of results for control samples Result of the analysis is considered reliable only if the results obtained for Positive and Negative Controls of amplification as well as Negative Control of extraction are correct Table Results for controls Stage for Control cantral Interpretation NCE DNA extraction Neg 36 Valid result NCA Amplification Neg Neg Valid result C Amplification lt 36 lt 34 Valid result Sacace MTB Real TM VER 21 03 2013 Analysis of result for clinical samples Table Interpretation of results for the samples
12. se 0 03 ml e UDG Enzyme 0 03 ml Contains reagents for 55 tests must be used in the isolation procedure as Negative Control of Extraction add 10 ul of Internal Control during the DNA isolation directly to the sample lysis mixture Sacace Real TM VER 21 03 2013 MATERIALS REQUIRED BUT NOT PROVIDED Zone 1 sample preparation e DNA extraction kit Module No 1 e Biological cabinet e Vortex e 65 C 2 5 dry heat block e Desktop microcentrifuge for eppendorf type tubes max 16 000 x g e Tube racks e Microcentrifuge tubes 1 5 2 0 ml e Pipettes with sterile RNase free filters tips e Biohazard waste container e Disposable gloves powderless e Refrigerator Freezer Zone 2 Real Time amplification e Real Time Thermalcycler e Tubes or PCR plate e Workstation e Pipettes with sterile RNase free filters tips e Tube racks STORAGE INSTRUCTIONS Store kit at 2 8 C Part N 3 MTB Real TM must be stored at 20 C The kit can be shipped at 2 8 C but should be stored at 2 8 C and 20 C immediately on receipt Store DNA RNA Prep kit at 2 25 C STABILITY MTB Real TM is stable up to the expiration date indicated on the kit label The product will maintain performance through the control date printed on the label Exposure to light heat or humidity may affect the shelf life of some of the kit components and should be avoided Repeated thawing and freezing of these reagents should b
13. se of this product should be limited to personnel trained in the techniques of DNA amplification EN375 Strict compliance with the user manual is required for optimal PCR results Attention should be paid to expiration dates printed on the box and labels of all components Do not use a kit after its expiration date SAMPLE COLLECTION STORAGE AND TRANSPORT Real TM can analyze DNA extracted from e Sputum bronchial or tracheal lavage must be treated with the following procedure o Collect sputum into 50 mL single use PP tubes with a screw cap o In a biological safety cabinet homogenize samples after mixing with equal volume of 496 NaOH solution N acetyl L cysteine may be added if required in the amount of 50 70 mg per sample Mix intensely with a tube rotator for 5 20 minutes depending on the density of a sample o Centrifuge samples at 3000 rpm 2800 3000 g for 15 min and carefully discard the supernatant leaving 500 1000 ul in the tube Resuspend sediment and transfer it into a 1 5 ml tube o Centrifuge samples at 12000 rpm for 5 10 min discard the supernatant and use the same 1 5 ml sample tube for DNA isolation from sample sediment e tissue 71 0 gr homogenized with mechanical homogenizer or scalpel glass sticks teflon pestles and dissolved in 1 0 ml of saline water or PBS sterile 1 volume of tissue to 1 volumes of saline solution Vortex vigorously and incubate 30 min at room temperature Transfer the supernatant into
14. tains UDG Enzyme which is added to the reaction mix Since deoxyuridine triphosphate dUTP is only present in amplicon while deoxythymidine triphosphate dTTP is present in MTB DNA the use of UDG enzyme degrades only amplicons generated from previous runs avoiding possibility of amplicon contamination UDG is active at room temperature during Mastermix preparation while during amplification is inactive not affecting the correct and wanted experiment s amplicon Sacace MTB Real TM VER 21 03 2013 MATERIALS PROVIDED Module No 1 Real Time PCR kit B15 50FRT Part 2 Controls e 8 IC 0 1 ml e Negative Control 1 2 ml e MTB IC 1 0 ml e DNA buffer 0 5 ml Part N 3 MTB Real TM Real Time amplification e PCR mix 1 2 x 0 28 ml e PCR Buffer Flu 0 28 ml TaqF Polymerase 0 03 ml e UDG Enzyme 0 03 ml Contains reagents for 55 tests Module No 2 Complete Real Time PCR test with DNA purification kit TB15 50FRT Part N 1 DNA RNA Prep Sample preparation e Lysis Sol 15 ml e Prec Sol 20 ml e Washing Sol 3 25 0 ml e Washing Sol 4 10 0 ml e RE buffer 4x 1 2 ml Contains reagents for 50 extractions Part 2 Controls e amp IC 0 1 ml e Negative Control C 1 2 ml e MTB IC 1 0 ml e DNA buffer 0 5 ml Part N 3 Real TM Real Time amplification e PCR mix 1 2 x 0 28 ml e PCR Buffer Flu 0 28 ml e TaqF Polymera
15. tive Control C to the tube labeled Vortex the tubes and incubate for 5 min at 65 Centrifuge for 7 10 sec 6 Add 400 ul of Prec Sol and mix by vortex Centrifuge all tubes at 13 000 r min for 5 min and 9 10 11 using a micropipette with a plugged aerosol barrier tip carefully remove and discard supernatant from each tube without disturbing the pellet Change tips between the tubes Add 500 pl of Wash Sol 3 into each tube Vortex vigorously to ensure pellet washing Centrifuge all tubes at 13 000 r min for 60 sec and using a micropipette with a plugged aerosol barrier tip carefully remove and discard supernatant from each tube without disturbing the pellet Change tips between the tubes Add 200 pl of Wash Sol 4 into each tube Vortex vigorously to ensure pellet washing Centrifuge all tubes at 13 000 r min for 60 sec and using a micropipette with a plugged aerosol barrier tip carefully remove and discard supernatant from each tube without disturbing the pellet Change tips between the tubes Incubate all tubes with open caps at 65 for 5 min Resuspend the pellet in 50 pl of RE buffer elution volume can be increased up to 90 ul Incubate for 5 min at 65 C and vortex periodically Centrifuge the tubes at 13000g for 60 sec The supernatant contains RNA DNA ready for amplification If amplification is not performed the same day of extraction the processed samples can be stored at 2 8 C for at maximum period
16. tuberculosis because their immune systems are compromised by immunosuppressive drugs substance abuse or HIV AIDS Early diagnosis of tuberculosis makes effective treatment possible and increases the probability of clinical outcome owing to quite effective antituberculosis therapy however the tuberculosis diagnosis has certain difficulties According to international standards tuberculosis diagnosis must be confirmed either by bacteriology or by histology studies but the bacteriological methods do not always allow to detect Mycobacterium tuberculosis in people affected with pulmonary tuberculosis and especially with extrapulmonary tuberculosis The application of molecular biology methods allow to overcome the difficulties in the diagnosis of Mycobacterium tuberculosis but due to the biological peculiarities of this microorganism and immune response of human organism tuberculosis can not be diagnosed only by one method Direct and indirect diagnostic methods are applied in phthisiology Smear bacterioscopy with Ziehl Neelsen stain technique is a rapid and cheap method but it has low sensitivity not high specificity and cannot differentiate TB from nontuberculous mycobacteria The diagnostic sensitivity of the method doesn t exceed 20 4096 Smear fluorescence microscopy is a more sensitive method requiring less enlargement of the microscope during the study and thus allowing to observe a larger area if compared to the standard microscopy with immers
17. vis strains 1 2 3 4 5 8 BCG 14 1414 AN 5 Mycobacterium africanum Mycobacterium microti The DNA concentration was 10 genomic equivalents ml While testing the kit MTB Real TM gives a positive results with all the strains belonging to Mycobacterium tuberculosis complex ANALYTICAL SENSITIVITY The analytical sensitivity of the MTB Real TM kit was valuated using the serially dilution of Standard DNA of the Mycobacterium tuberculosis and QCMD MTB Panels 2003 2009 2010 The analytical sensitivity of the kit MTB Real TM was not less than 5 CFU sample Target region IS 6110 Sacace Real TM VER 21 03 2013 TROUBLESHOOTING 1 Weak Ct gt 36 or no signal of the IC Joe Hex Cy3 channel for the Negative Control of extraction e The PCR was inhibited Make sure that you use a recommended DNA extraction method and follow to the manufacturer s instructions Re centrifuge all the tubes before pipetting of the extracted DNA for 2 min at maximum speed 12000 16000 9 and take carefully supernatant Don t disturb the pellet sorbent inhibit reaction e The reagents storage conditions didn t comply with the instructions Check the storage conditions The PCR conditions didn t comply with the instructions Check the PCR conditions and select for the IC detection the fluorescence channel reported in the protocol e The IC was not added to the sample during the pipetting of reagents Make attention during the
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