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MTBC Detection Kit v1 USER MANUAL

1. positive control or kit components Follow the instructions for the storage of kit components Section 3 Storage ternal control Deterioration of the internal control or detection mix 2 Follow the instructions for the storage of kit components See Section 3 Storage PCR inhibition Make sure that you use the recommended DNA isolation method See 9 3 DNA isolation DNA lost during isolation Make sure that you use the recommended DNA isolation method See 9 3 DNA isolation Signal from FAM Filter in the Negative Control Contamination Use filter tips Repeat PCR with new kit components The Threshold is Above Low Signals Uneven Traces Code MBO5v2f Date July 2011 The threshold should be manually adjusted Using the mouse pull the threshold down until it cuts the low signals Avoid the background and the signal from negative control Baseline cycles should be readjusted Assign new baseline cycles manually and recalculate threshold cycles 12 SPECIFICATIONS 12 1 Sensitivity Analytical sensitivity may be expressed as the limit of detection i e the smallest amount of the target marker that can be precisely detected The detection limit of an individual analytical procedure is the lowest amount of nucleic acid in a sample which can be detected but not necessarily quantitated as an exact value The analytical sensitivity or detection limit for NAT assays i
2. should be stored separately to avoid contact with the kit components e Pathogen information should be reviewed to be aware of the health related risks e Clinical samples should be handled with extreme caution Physical contact with pathogens should be avoided by wearing lab coats and gloves no allowance for eating or drinking within the workspace prevention of unauthorized individuals access to the working area e All the pathogenic wastes produced during the nucleic acid isolation step including the biological samples and material contacted with them should be discarded into medical waste and disposed safely 6 PRODUCT USE LIMITATIONS e All the components may exclusively be used for in vitro diagnostics e This product should be used in accordance with this user manual e This product is to be used by personnel specially trained to perform in vitro diagnostic procedures 7 PATHOGEN Causative Agents MTBC Mycobacterium Tuberculosis Complex contains seven closely related bacteria species with distinct host tropisms including M tuberculosis M bovis M bovis BCG M africanum M canetti M caprae M microti and M Pinnipedi The most common member of the complex is the M Tuberculosis The human pathogens M africanum and M canettii and species adapted to animals M bovis bovine M caprae goats M pinnipedii seals and M microti rodents are the other members of the complex Members of the MTBC are estimated as gene
3. to avoid exogenous contamination with mycobacterium All steps of DNA extraction must be performed under a biological safety cabinet In order to ensure safety samples might be processed in a decontamination procedure if desired On the other hand decontamination procedures might reduce the sensitivity of the assay Specimen should be stored at 4 C for short term 24 hours if it is to be processed in a day or frozen at 20 C for longer periods of time Sensitivity of the PCR might be reduced because of repeated cycles of freeze thaw DNase RNAse free polypropylene tubes must be used for storage The samples should be transported in containers with capacity to resist pressure Transportation should be done according to local and national regulations for pathogen material transport 9 2 Interfering Substances To avoid possible influences on PCR e Samples which have been collected in waxed containers or in propylene glycol test tubes e Food particles or mouthwash e Decontamination reagents such as isoniazid might reduce PCR sensitivity must not be used 9 3 DNA Isolation We recommend that the Magnesia 16 Nucleic Acid Extraction System Magnesia Bacterial DNA Extraction Kit Anatolia Geneworks isolation system or an alternative high quality DNA extraction system is used with Bosphore MTBC Detection Kit v1 The DNA isolation should be performed according to the manufacturers instructions The starting volume is 200 ul the elution
4. volume is 100 ul 9 4 Kit Components 9 4 1 PCR Mix HotStarTaq DNA Polymerase HotStarTaq DNA Polymerase is a modified form of a recombinant 94 kDa DNA polymerase originally isolated from Thermus aquaticus cloned into E Coli The enzyme is provided in an inactive form It is activated by a 15 minute 95 C incubation step This prevents the formation of misprimed products and primer dimers during reaction setup and the first denaturation step leading to high PCR specificity and accurate qualitative analysis QuantiTect Probe PCR Buffer contains Tris Cl KCI NH4 2SO4 8 mM MgCls pH 8 7 20 C dNTP Mix Contains ultrapure quality dATP dGTP dCTP ve dTTP dUTP 9 4 2 Detection Mix 1 Detection Mix 1 contains MTBC specific forward and reverse primers and a dual labeled probe 9 4 3 Detection Mix 2 Detection Mix 2 contains internal control specific forward and reverse primers and a dual labeled probe 9 4 4 Internal Control An internal control is included in the kit to control PCR inhibition The internal control is a synthetic DNA molecule derived from human genome It is added directly into the PCR master mix to control the PCR inhibition exclusively For this purpose 0 2 ul of internal control should be added for each reaction into the master mix Lack of Code MBO5v2f 4 Date July 2011 internal control amplification in the FAM negative samples may indicate a problem in PCR or PCR inhibition In this case PCR should be repeate
5. 000 p 653 655 2 Anonymous Tuberculosis Fact Sheet 2009 Update World Health Organization 3 Thierry Zozio Caroline Allix Selami Gunal Genotyping of Mycobacterium tuberculosis clinical isolates in two cities of Turkey Description of a new family of genotypes that is phylogeographically specific for Asia Minor BMC Microbiol 2005 5 44 4 Barry R Bloom William N Rom Stuart M Garay Tuberculosis Pathogenesis Protection and Control Oct 1 1994 p 47 49 14 SYMBOLS Use by Lot Batch Catalog number Temperature limitation Caution consult accompanying documents Manufacturer E gt gt fi i IVD In Vitro Diagnostic Medical Device 15 CONTACT INFORMATION Anatolia Geneworks Egitim Mh Kasap Ismail Sk No 10 23 Kadikoy 34722 ISTANBUL TURKEY Phone 90 216 330 04 55 Fax 90 216 330 00 42 E mail info anatoliageneworks com www anatoliageneworks com Registered Trademarks Anatolia Geneworks Montania Magnesia and Bosphore are registered trademarks of Anatolia Tani ve Biyoteknoloji A S Code MBO5v2f 11 Date July 2011
6. GeneK LineGene 9600 Bioer Rotorgene 2000 3000 6000 Q Qiagen e 0 2 ml Thin Wall PCR tubes or strips e Magnesia 16 Nucleic Acid Extraction System Magnesia Bacterial DNA Extraction Kit Anatolia Geneworks or other high quality DNA extraction kits and systems e Deep freezer 20 C e Desktop centrifuge with rotor for 2 ml microcentrifuge tubes e Calibrated adjustable micropipettes e DNAse RNAse pyrogen free micropipette tips with filters e DNAse RNAse pyrogen free 1 5 or 2 ml microcentrifuge tubes e Disposable laboratory gloves Code MBO5v2f 1 Date July 2011 5 IMPORTANT NOTES AND SAFETY INSTRUCTIONS Important e The product should be delivered on dry ice Check for presence of dry ice upon arrival e Check for the expiry dates on the box and tube labels upon arrival Do not use expired products or components e Calibrated or verified micropipettes DNAse RNAse pyrogen free micropipette tips with filters and DNAse RNAse pyrogen free microcentrifuge tubes should be used e Before starting a test procedure all components should be thoroughly thawed After thawing all components should be centrifuged briefly spin down for 3 5 seconds and mixed well to ensure homogeneity prior to use e The kit components should be kept on ice or a cooling block until the reaction is prepared and they should be quickly returned to 20 C e PCR and nucleic acid isolation must be performed in different compartments Samples
7. MTBC Detection Kit v1 USER MANUAL For in vitro Diagnostic Use Document Code MBO5v2f Approval Date July 2011 10 11 12 13 14 15 Contents Product Description Content Storage Required Materials and Devices Important Notes and Safety Instructions Product Use Limitations Pathogen Method Procedure 9 1 Sample Preparation Storage and Transport 9 2 Interfering Substances 9 3 DNA Isolation 9 4 Kit Components 9 4 1 PCR Mix 9 4 2 Detection Mix 1 9 4 3 Detection Mix 2 9 4 4 Internal Control 9 4 5 Positive Control 9 5 Preparing the PCR 9 6 Programming the Montania 483 Real Time PCR Instrument Analysis Troubleshooting Specifications 12 1 Sensitivity 12 2 Cross Reactivity 12 3 Reproducibility and Precision References Symbols Contact Information Code MBO5v2f Date July 2011 10 10 10 10 11 11 11 1 PRODUCT DESCRIPTION Bosphore MTBC Detection Kit v1 detects MTBC DNA in human biological samples encompassing all the complex MTBC genotypes M tuberculosis M bovis M bovis BCG M africanum M canetti M caprae M microti and M Pinnipedi The analytic sensitivity is 128 copies ml A region within the 16S ribosomal RNA gene is amplified and fluorescence detection is accomplished using the FAM filter An internal control has been integrated into the kit in order to check PCR inhibition The amplification data of the internal control is detected with the Cy5 filt
8. Real Time PCR method Polymerase chain reaction is a technique that is used for amplification of a DNA region The reaction occurs by the repeating cycles of heating and cooling The main components of PCR are primers dNTPs Taq polymerase enzyme buffer solution and template As a brief explanation primers are small synthetic DNA those anneal to the specific regions of the template in order to start the synthesis dNTPs are the building blocks of the amplified products Taq polymerase amplifies the DNA template Buffer solution provides the pH adjustment required for the reaction and template as referred is the target region for synthesis In Real Time PCR technique in contrast to conventional PCR PCR product can be monitored during the reaction Therefore Real Time PCR obviates the need for further analysis methods like gel electrophoresis whereby minimizing the risk of contamination Dual labeled probes employed in the reaction in addition to the conventional PCR reagents enable detection of the amplified target with increased sensitivity The assay utilizes the 5 exonuclease activity of Taq Polymerase to cleave a dual labeled fluorescent hybridization probe during the extension phase of PCR The probe is labeled at the 5 end with a fluorescent reporter molecule and at the 3 end with another fluorescent molecule that acts as a quencher for the reporter When the two fluorophores are in close proximity and the reporter i
9. S By the end of the thermal protocol the Montania 483 Real Time PCR Instrument software automatically calculates the baseline cycles and the threshold Example of an amplification curve is given in Fig 4 Code MBO5v2f 7 Date July 2011 FAAP ERE Sotnas ek Anarsia ToT Heb sas 0 gt B2123 07 Module Edit i _ TestResult Module Mode An ation Curve I Standard urve T Report Mode ji 143180 120671 ss pi We 1933 asson anses s7278 aass7 0 28636 anng 0 00000 Fae a M e o M o anma 2 lplel gt 2 Fig 4 Amplification Curve of a Bosphore MTBC v1 test Analysis of the results should be performed by trained personnel who have received the required training for analysing Real Time PCR data We recommend that the test results must be evaluated by an expert clinician taking the patient s clinical findings and the results of other tests into consideration All analysis is done automatically in routine use However when the trained personnel who have received the required training from manufacturer consider it as necessary the system allows pulling down the threshold as much as possible in order to detect low positive samples In this case attention should be paid to keep the threshold line above the background and to keep the correlation coefficient at the maximum possible value and within its acceptance criteria Positive control of Bosphore MTBC v 1 is essential for accurate re
10. d In samples that contain a high bacterial load internal control can be suppressed and no increase of the signal is detected Please use the table below for the interpretation of internal control data MTBC FAM Internal Control Cy5 Interpretation Sample positive Sample negative Sample positive Repeat the test 9 4 5 Positive Control The positive control contains MTBC DNA It can be included in the PCR to test the efficiency of the PCR exclusively The threshold cycle for the positive control is given in the acceptance criteria table Section 10 Analysis Threshold cycles higher than the acceptance criteria may indicate an efficiency loss in the reaction 9 5 Preparing the PCR Positive control should be added into the PCR reaction together with the samples and the negative control PCR grade water in order to deduce the positivity negativity of the samples Make sure that all the kit components are thawed before use Refer to the table below for preparing the PCR It is for only one reaction multiply these values with the sample number to find the values required for the master mix While preparing master mixes for more than 5 samples an extra 10 should be added to the total sample number PCR Mix 20 ul Detection Mix 1 1 6 ul Detection Mix 2 1 2 ul Internal Control 0 2 ul Sample DNA 18 ul Negative Positive Control Total Volume 41 0 ul Pipette 23 pl of the master mix into th
11. e PCR tubes or strips and add 18 ul of DNA sample positive or negative control Close the tube cap Make sure that the solution in each tube is at the bottom of the tube Centrifuge if necessary 9 6 Programming the Montania 483 Real Time PCR Instrument The thermal protocol for Bosphore MTBC Detection Kit v1 is composed of an initial denaturation for activation the HotStarTaq DNA Polymerase a two step amplification cycle and a terminal hold The real time data is collected at the second step of the amplification cycle Initial denaturation 95 C 14 30 min Denaturation 97 C 00 30 min Annealing and Synthesis 53 C 01 30 min 50 cycles Data Collection Hold 22 C 05 00 Montania 483 Real Time PCR Instrument is installed and calibrated as it is delivered to the end user In order to establish an appropriate link between the system components first the thermal cycler and the optical module and then the PC and the software should be started Before starting a Real Time PCR reaction using the Bosphore Kits the following steps should be completed e Choose the filter pairs to be used FAM and Cy5 Code MBO5v2f 5 Date July 2011 e Identify unknown samples positive and negative controls e Select the correct thermal protocol These steps are described below From the main menu of the Montania 483 Real Time PCR Instrument File and then New is selected Create a new Experiment is selected In the Se
12. er The internal control is added during PCR step 2 CONTENT Bosphore MTBC Detection Kit v1 is composed of Real Time PCR reagents an internal control and a positive control Component REAGENT 100 Tests 50 Tests 25 Tests 1 dH20 1000 ul 1000 ul 500 ul 2 PCR Mix 2240 ul 1120 pl 560 ul 3 Detection Mix1 176 ul 88 ul 44 ul 4 Detection Mix2 132 ul 66 ul 33ul 5 Internal Control 23 ul 15 ul 15 ul 6 Positive Control 1 160 ul 80 ul 40 ul 3 STORAGE Bosphore MTBC Detection Kit v1 PCR reagents should be stored at 20 C Repeated thawing and freezing gt 3x should be avoided since it may reduce sensitivity If the components are to be used in small amounts they should be frozen in aliquots While preparing the PCR the components should not be exposed to room temperature for more than 10 min and the detection mix components should not be exposed to light more than 1 2 min We recommend preparing the PCR on a cooling block and keeping the detection mixes within a closed container The components maintain their stability until the expiry dates on the labels if they are stored at advised conditions 4 REQUIRED MATERIALS AND DEVICES e Montania 483 Real Time PCR Instrument Anatolia Geneworks or another Real Time PCR system with FAM and Cy5 filters iCycler iQ5 CFX BioRad LightCycler 1 5 2 0 480 Roche 7500 Real Time PCR System ABI Stratagene Mx3005P Mx3000P Agilent Line
13. lect Channel window channels 1 FAM and 3 Cy5 are selected Fig 1 Samples positive and negative controls are identified in the Module Edit menu Fig 2 To select the thermal protocol Gene Amplification menu is used The Open button in the Experiment Program is clicked and the appropriate thermal protocol is selected Fig 3a The thermal cycles of the selected protocol is displayed The experiment starts by clicking the Start button Fig 3b Ostia vx Ga E Fig 1 Filter Selection in Montania 483 _File F _Ecit E _Settings S _View V _Analysis A _Toots T _Help H Coe Hols Ox esSia2isie Module Edit Gene Amplification Test Result 1 2 fi a 4 E i 6 I 7 a s 10 I m 12 B rac Tac rac Tac mic rac Sample Sample Sample Sample Sample Sample ic Ic Ic Sample Sample Sample Sample Sample Sample c Standard cHI Sample Negative Positive e Control CH3 Blank Template Editon E Patient Information Fig 2 Sample Location and Identification Code MBO5v2f 6 Date July 2011 File F Edit E Settings S _View V _Analysis A Tools 1 _Help H DSHA xE Sila 2ale Module Edit Gene Amplification Test Result Amplification Program if Temperature Curve T fale Give Eterna I Block Tempsatue C 0 TF Sample TemperatweT 0 F Hot Lid Temperatue C 0 Punning Status Punning D zenle Yeni klaso
14. ositive 3 Sample Ic 3676 No Result as 1 Sample 1B 35 67 Positive 3 Sample Ic 3760 No Result ag 1 Sempe TB 25 19 Positive 3 Sample IC NoCt No Result ao Sample TB 28 73 Positive 3 Sample Ic NoCt No Result ag 1 Negative 1B NoCt Negative 3 Negative Ic 35 99 No Result ag 1 Negative 1B NoCt Negative 3 Negative Ic 36 65 No Result ca 1 2 3 4 5 7 s p 10 at 12 a 2 i E ji f Fig 6 A Report Mode Screen Showing the Results The following table shows the possible results and their interpretation Signal detected in FAM filter pair No need to check the internal control since the sample is positive high positive samples may suppress the signal from the internal control The sample contains MTBC DNA the result is positive No signal in FAM signal in Cy5 The MTBC DNA in the sample is not detectable Signal from Cy5 filter pair rules out the possibility of PCR inhibition No signal in FAM and Cy5 The diagnosis is inconclusive No signal in Cy5 points out to PCR inhibition or to a problem in DNA isolation See 11 Troubleshooting 11 TROUBLESHOOTING Please contact the manufacturer in case of a problem during a run Late or no signal from the FAM filter from the positive control No signal from the in Wrong thermal protocol is chosen Make sure that the correct thermal protocol is chosen Deterioration of the positive control Don t use expired
15. r Cycle times o Asiti fel M De i tirme tarihi Temperate 0 TB Kargidan Vakiem A Fv6Sprg Holdratme 000 EE Maseusta B HBVL pro Running tine 000000 E Son Yerler E HPV pig 14K prg 02052011 1113 Dosyas Contal Mode G Kitapliklar B MTuberculosis53 pro 1905201 Ha With Hot Lid E Belgeler L TEST prg 23 01 2007 07 23 PRG Dosyas Block Control ad M zik E Resimler H video Experirent Program Open Delete 1 Bilgisayar New Edt Anatolia 2 C a Anatolia 2 Backu 4 r T z E Dosya Ade MTuberculosis53 prg Fig 3a Selecting the Thermal Protocol File F _Edit E Oe ao 9 View V Analysis A Tools T Help H xI a el Module Edit Gene Amplification Test Result Ampification Program L Temperature Curve T Real Time Curve Temperature Moriter C Program Fles SLAN Real Time PCR Detection System PEOGRAM MT ubsrculosi6 org Block Terperatue C 0 7 Sample Temperature C 0 Hot Lid Temperature 0 groe __sge Gren j Rumning Status Punning Ciele imes a Temperate 0 Holding tine www Rurring tine 00000 Corttl Mace Wih Hoe Lid Block Contol Experiment Program Flucrescence Test Those Dette ten Edt Rawdata Nomalzed Dat Start zoc mam Segnent1x1 Segment 2x50 Segment 3x1 Fig 3b Starting the Experiment 10 ANALYSI
16. s excited by light no reporter fluorescence can be detected During the elongation step of PCR Taq Polymerase encounters and cleaves the probe bound to the template As the reporter is freed from the suppressing effect of the quencher fluorescence signal can be detected The fluorescence generated by the reporter increases as the PCR product is accumulated the point at which the signal rises above background level and becomes distinguishable is called the threshold cycle C There is a linear relationship between the log of the starting amount of a template and its threshold cycle Bosphore MTBC Detection Kit v1 employs multiplex PCR and an internal control is incorporated into the system in order to check for possible PCR inhibition MTBC DNA and an internal control are co amplified in a single reaction using sequence specific primers The fluorescent signal generated by the MTBC amplification is detected by a probe labeled at the 3 end with FAM through the FAM channel The fluorescent signal generated by the internal control amplification is detected by a second probe labeled at the 5 end with a different reporter molecule Cy5 through the Cy5 channel Code MBO5v2f 3 Date July 2011 9 PROCEDURE 9 1 Sample Preparation Storage and Transport Human respiratory samples such as bronchoalveolar lavage BAL sputum bronchial mucosa should be collected To isolate bacterial DNA from the clinical specimen great care should be taken
17. s expressed by the 95 positive cut off value The analytical detection limit for Bosphore MTBC v1 was found to be 1 28x10 copies ml p 0 05 The sensitivity was determined using a dilution series of a quantitated DNA Control The dilutions were tested in different runs in replicates The results were analyzed by probit method 12 2 Cross Reactivity To eliminate potential cross reactivity both assay design evidence and experimental studies were employed Primer and probe sequences were checked for possible homology to other known pathogen sequences by sequence comparison analysis using database alignment Samples of Chlamydia Toxoplasma CMV EBV Parvovirus B19 BKV with known high positivity were tested and found negative 12 3 Reproducibility Reproducibility data on C value basis were obtained by the analysis of positive control of the Bosphore MTBC Detection Kit v1 Test was performed in at least 4 replicates by 3 different operators on multiple days using 3 different lots The resulting data is given in Table 1 Table 1 Reproducibility Data MTBC Standard Variance Coefficient of 107 copies ml deviation variation Intra assay Variability N 4 Inter lot Variability N 3 Inter operator Variability N 3 Total Inter assay Variability N 5 Code MBO5v2f 10 Date July 2011 13 REFERENCES 1 K E Nelson C Williams and N Graham Infectious Disease Epidemiology Theory and Practice July 15 2
18. sult analysis The cycle threshold acceptance criteria for the positive control is 33 4 Test results should not be reported unless the assay results meet the criteria stated above Please contact the manufacturer if impairment in the product s performance is observed See the last page for contact information The qualitative results of the test are displayed on the Report Mode screen The samples that cross the threshold in Channel 1 FAM are displayed as positive whereas samples that do not cut the threshold are displayed as Negative or No Ct These samples are regarded as negative or having a bacterial load below the detection limit of the assay For these undetectable samples the Cy5 data of the internal control should also be checked to avoid false negative results Fig 6 Code MBO5v2f 8 Date July 2011 ae a ial t Lianan ae Aidaa iil a ad Ce eolrOx Osiaais lki Module Edit Gene Amplification Test Result Module Mode i Amplification Curve Standard Curve Report Mode Melting Curve Well Channel Type Testitem Property CT TestResuk Label Name Age CaseID BediD Inpatient O Outpatient ID Sample Type Sample Date Diagnosis Doctor Detect Date Otice Experimenter Assessor Remark a Sme TB 26 55 Positive 3 Sampe IC NoCt NoResut a 1 Sample TB 30 33 Positive 3 Sample Ic NoCt No Result A4 1 Sample 1B 33 91 P
19. tically monomorphic with a high level of genomic sequence similarity gt 99 95 limited horizontal gene transfer and a clonal population structure It is accepted as the genetic diversity among MTBC strains has not any clinical significance because of this apparent homogeneity They cause tuberculosis in humans by localizing in the lungs 1 Code MBO5v2f 2 Date July 2011 Epidemiology Tuberculosis is the most common second infection worldwide causing the death of young and older people One third of the world population is infected with tuberculosis Each year 9 million people worldwide have been infected with tuberculosis and nearly 2 million of this has resulted in death In Turkey between 10 million and 20 million people are infected with tuberculosis and carry infectious agent of tuberculosis mycobacterium tuberculosis The prevalence of TB is approximately 27 in one hundred thousand 2 3 Modes of Transmission Even though it can affect all organs of the body the main entry of the infection is respiratory system through lung It is transmitted through air droplets which are generated when TB infected persons cough sneeze speak etc Also being in close contact with a person that has active TB disease with TB germs present in the sputum and consumption of unpasteurized infected cow s milk that has Mycobacterium bovis are the other causes of the human tuberculosis 4 8 METHOD Bosphore MTBC Detection Kit v1 is based on the

MTBC Detection Kit v1 USER MANUAL

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