MTB Real TM Diff PCR ver 24062013 - bio
Contents
1. 0 5 ml e Negative Control C 1 2 ml e MTBIC 0 5 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 purification procedure directly to the sample lysis mixture Sacace MTB Diff Real TM VER 24 06 2013 MATERIALS REQUIRED BUT NOT PROVIDED Zone 1 sample preparation e DNA extraction kit Module No 1 e Biological cabinet e Desktop microcentrifuge for eppendorf type tubes e Dry heat block e Vortex mixer e Pipettes e Sterile pipette tips with filters e 1 5 ml polypropylene sterile tubes e Biohazard waste container e Refrigerator Freezer Zone 2 Real Time amplification e Real Time Thermal cycler e Reaction tubes e Workstation e Pipettes adjustable e Sterile pipette tips with filters e Vortex mixer e Freezer refrigerator STORAGE INSTRUCTIONS Store kit at 2 8 C PCR mix 1 and TaqF Polymerase 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 Sorb B kit at 2 25 C STABILITY MTB Diff 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 should2. be 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 Diff Real TM VER 24 06 2013 WARNINGS AND PRECAUTIONS The user should always pay attention to the following gt A 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 e 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 e Use disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterwards e Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas e Do not use a kit after its expiration date e Dispose of all specimens and unused reagents in accordance with local authorities
3. regulations e Specimens should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices e Clean and disinfect all sample or reagent spills using a disinfectant such as 0 5 sodium hypochlorite or other suitable disinfectant e 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 e Material Safety Data Sheets MSDS are available on request e 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 Diff Real TM VER 24 06 2013 PRODUCT USE LIMITATIONS Use 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 STORA
4. GE AND TRANSPORT MTB Diff Real TM can analyze DNA extracted with DNA Sorb B REF K 1 1 B 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 Ina biological safety cabinet homogenize samples after mixing with equal volume of 4 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 1 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 a new 1 5 ml tube e whole blood collected in either ACD or EDTA tubes e liquor stored in Eppendorf tube e feces prepare 20 feces suspension by adding in 5 ml tube of 4ml of Saline Solution and 1 0 gr approx 1 0 ml of feces Vortex to get the homogeneous
5. _ sacace BIOTECHNOLOGIES MTB Diff Real TM Handbook Real Time Amplification test for the qualitative detection and differentiation of M tuberculosis M bovis and M bovis BCG REF B41 50FRT REF TB41 50FRT 2 50 Sacace MTB Diff Real TM VER 24 06 2013 NAME MTB Diff 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 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 tub
6. amples to the appropriate tube Prepare Controls as follows add 100 ul of C Neg Control provided with the amplification kit to the tube labeled Crneg 5 Vortex the tubes and incubate for 5 min at 65 C Centrifuge for 5 7 sec If the sample is not completely dissolved it is recommended to re centrifuge the tube for 5 min at a maximum speed 12000 16000 g and transfer the supernatant into a new tube for DNA extraction 6 Vortex vigorously Sorbent and add 25 ul to each tube 7 Vortex for 5 7 sec and incubate all tubes for 3 min at room temperature Repeat this step 8 Centrifuge all tubes for 30 sec at 5000g 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 9 Add 300 pl of Washing Solution 1 to each tube Vortex very vigorously and centrifuge for 30 sec at 5000g Remove and discard supernatant from each tube 10 Add 500 ul of Washing Solution 2 to each tube Vortex vigorously and centrifuge for 30 sec at 10000g Remove and discard supernatant from each tube 11 Repeat step 10 and incubate all tubes with open cap for 5 10 min at 65 C 12 Resuspend the pellet in 50 ul of DNA eluent Incubate for 5 min at 65 C and vortex periodically 13 Centrifuge the tubes for 1 min at maximum speed 12000 16000 g The supernatant contains DNA ready for amplification If amplification is not performed in the same day of e
7. erculosis 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 INTENDED USE The development of test to differentiate between infection with Mycobacterium tuberculosis or Mycobacterium bovis and vaccination with M bovis BCG could greatly assist in the diagnosis of early infection as well as enhance the use of tuberculosis vaccines on a wider scale Sacace MTB Diff Real TM VER 24 06 2013 PRINCIPLE OF ASSAY kit MTB Diff Real TM is a Real Time Amplification test for the qualitative detection and differentiation of M tuberculosis M bovis and M bovis BCG in the sputum urine blood bronchial lavages tissue and other biological materials DNA is extracted from samples amplified using Real Time Amplification and detected using fluorescent reporter dye probes specific for M tuberculosis M bovis M bovis BCG and IC M tuberculosis C is DNA fragment of IS 6110 insertion of Mycobacterium tuberculosis modified and cloned in bacteriophage A containing DNA fragmen
8. he manual 3 Fam Green Joe Yellow HEX TET Cy3 and Rox Orange TexasRed signal with Negative Control of extraction e 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 e 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 MTB Diff Real TM VER 24 06 2013 KEY TO SYMBOLS USED List Number Caution Contains sufficient LO Lot Number for lt n gt tests Expiration Date VER Version Negative Control of m E amp E Store at NCA P Amplification Negative control of Manufacturer NCE sally Extraction Consult instructions C Positive Control of for use Amplification For Research Use IC Internal Control RUO Only SaCycler is a registered trademark of Sacace Biotechnologies iQ5 is a registered trademark of Bio Rad Laboratories Rotor Gene Technology is a registered trademark of Qiagen MX3005P is a registered trademark of Stratagene ABI is a registe
9. or every run to verify that the specimen preparation the amplification and the detection steps are performed correctly If the controls are out of their expected range see table Results for Controls all of the specimens and controls from that run must be processed beginning from the sample preparation step Sacace MTB Diff Real TM VER 24 06 2013 TROUBLESHOOTING 1 Weak or no signal of the IC Cy5 Red 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 g 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 e Improper DNA extraction Repeat analysis starting from the DNA extraction stage e 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 DNA extraction procedure 2 Weak 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 t
10. red trademark of Applied Biosystems SmartCycler is a registered trademark of Cepheid EcoqPCR is a registered trademark of Illumina A 7 Sacace Biotechnologies Srl 7 g via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com CERTIFICATO 9001 2008 Sacace MTB Diff Real TM VER 24 06 2013
11. resence of crossing of fluorescence curve with the threshold line M tuberculosis is detected on the Fam Green channel M bovis and M bovis BCG on the Joe Yellow HEX TET Cy3 channel M bovis BCG on the Rox Orange TexasRed channel and IC on the Cy5 Red channel Results are accepted as relevant if positive and negative controls of amplification and extraction are passed see table 1 Table 1 Results for controls Control SUES ah Interpretation control NCE DNA isolation NEG NEG NEG POS Valid result NCA Amplification NEG NEG NEG NEG Valid result MTB Diff C Amplification POS POS POS NEG Valid result The following results are possible Interpretation n P p ee Mix M tuberculosis M bovis BCG M bovis Mix M tuberculosis M bovis Mix M tuberculosis M bovis BCG M tuberculosis M bovis BCG M bovis M bovis BCG M bovis Negative Non valid QUALITY CONTROL PROCEDURE A defined quantity of Internal Control IC is introduced into each sample and control at the beginning of sample preparation procedure in order to control the extraction process of each individual sample and to identify possible reaction inhibition A negative control of extraction NCE negative amplification control NCA positive amplification control C are required f
12. suspension and centrifuge for 5 min to 7000 12000g and using a micropipette with a plugged aerosol barrier tip transfer in a new sterile 1 5 ml tube 100 ul of the supernatant e sinovial liquid stored in Eppendorf tube e urine sediment use the intermedium part of stream e prostatic liquid stored in Eppendorf tube 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 C Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents Sacace MIB Diff Real TM VER 24 06 2013 DNA ISOLATION The following kits are recommended gt DNA Sorb B Sacace REF K 1 1 B 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 MTB IC during DNA isolation procedure directly to the sample lysis mixture SPECIMEN AND REAGENT PREPARATION 1 Lysis Solution and Washing Solution in case of their storage at 2 8 C should be warmed up to 60 65 C until disappearance of ice crystals Prepare required quantity of 1 5 ml polypropylene tubes including one tube for Negative Control of Extraction 2 Add to each tube 10 ul of MTB IC Internal Control and 300 ul of Lysis Solution 3 Add 100 ul of S
13. ts 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 M tuberculosis is detected on the Fam Green channel M bovis and M bovis BCG on the Joe Yellow HEX TET Cy3 channel M bovis BCG on the Rox Orange TexasRed channel and IC on the Cy5 Red channel Sacace MTB Diff Real TM VER 24 06 2013 MATERIALS PROVIDED Module No 1 Real Time PCR kit B41 50FRT Part N 2 MTB Diff Real TM Real Time amplification e PCR mix 1 Diff 2 x 0 3 ml e PCR Buffer Flu 2 x 0 2 ml e TaqF Polymerase 0 03 ml e MTB Diff C 0 1 ml e DNA buffer 0 5 ml e Negative Control C 1 2 ml e MTBIC 0 5 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 purification procedure directly to the sample lysis mixture Module No 2 Complete Real Time PCR test with DNA purification kit TB41 50FRT Part N 1 DNA Sorb B Sample preparation e Lysis Solution 15 ml e Washing Solution 1 30 ml e Washing Solution 2 50 ml e Sorbent 1 25 ml e DNA eluent 5 ml Contains reagents for 50 extractions Part N 2 MTB Diff Real TM Real Time amplification e PCR mix 1 Diff 2 x 0 3 ml e PCR Buffer Flu 2 x 0 2 ml e TaqF Polymerase 0 03 ml e MTB Diff C 0 1 ml e DNA buffer
14. xtraction the processed samples can be stored at 2 8 C for at maximum period of 5 days or frozen at 207 80 C Sacace MTB Diff Real TM VER 24 06 2013 REAL TIME PCR 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 and 0 5 N 1 pl of TaqF DNA Polymerase Vortex and centrifuge briefly 3 Add to each tube 15 pl of Reaction Mix 4 Add 10 ul of extracted DNA to appropriate tube Prepare for each panel 2 controls e add 10 ul of DNA buffer to the tube labeled Amplification Negative Control e add 10 ul of MTB Diff C 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 Real Time PCR instruments Step Temperature C Time Cycles Hold 95 15 min 1 95 30s Cycling 65 30 s detection 45 72 30 s SaCycler 96 Sacace Rotor Gene 6000 Q Corbett Research Qiagen iQ5 BioRad Mx3005P Stratagene ABI 7500 Real Time PCR Applied Biosystems SmartCycler Cepheid EcogPCR Illumina Fluorescence is detected at 65 C in FAM Green JOE Yellow HEX Cy3 ROX Orange Texas Red and Cy5 Red fluorescence channels Sacace MTB Diff Real TM VER 24 06 2013 RESULTS ANALYSIS The results are interpreted through the p
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