3B BIOTUB QT Kit QT Kit - 3B BlackBio Biotech India Limited
Contents
1. AM A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools Labs S A Madrid Spain INTENDED USE 3 PRINCIPLE 3 REAGENTS 4 3B MTB NTM REAL TIME KIT 4 MTB DNA EXTRACTION KIT 5 INSTRUCTIONS FOR USE 6 Sample collection 6 Sputum samples 6 Tissues 6 Urine 6 Aseptic fluids body fluid bone marrow blood Bronchial washing 6 Stool 7 Preparation of Samples 8 Viscous Fluids sputum body fluids Pus 8 Non viscous Fluids aspiration body fluids urine pleural fluid 8 Fresh tissue Biopsy Endometrium tissue 9 Paraffin embedded tissue 10 Stool 10 Blood Bone marrow aspiration Menstrual blood 10 Broncho Alveolar Lavage BAL 11 DNA Extraction Step 12 REAL TIME PCR PROTOCOL FOR MTB NTM DETECTION 15 RESULT ANALYSIS 16 SPECIFICATIONS 18 TROUBLESHOOTING 21 STORAGE AND HANDLING amp ADDITIONAL REQUIREMENTS 23 GENERAL PRECAUTIONS 24 REFERENCES 25 2 Page 3B BIOTUB QT Kit www 3bblackbio com 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain INTENDED USE MTB NTM KIT accurately differentiates Mycobacterium tuberculosis MTB from non tuberculosis Mycobacterium species NTM in a qualitative quantitative form from various sources of clinical samples using Real time PCR PRINCIPLE 3B MTB NTM detection is a Real Time Amplification test for the qualitative quantitative detection of Mycobacterium tuberculosis compl2. Respiratory specimens sputum BAL BW tracheal aspirate etc 5 10 mL 1 Blood bone marrow 5 1 CSF 5 15 mL 1 Abscess wound aspirates 5 15 mL 1 fluids aspirates 5 15 mL dome Urine 30 50mL 10 mL Stool 2 58 1gm Tissue 5 Visible Pus 1 10 mL 1mL 7 Page 3B BIOTUB QT Kit www 3bblackbio com 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain B Preparation of Samples Prepare 2 N Acetyl Cystein NaOH Dissolve 2 g of NaOH 1 45 g of Sodium citrate 0 5 g N acetyl cystein in 100 ml of sterile distilled water Prepare 0 067M phosphate buffer solution by dissolving 2 37 g of Dibasic Sodium Phosphate Na2HPO 2 27 g Monobasic Potassium Phosphate in 500 ml of sterile distilled water Adjust the final pH to 6 8 i Viscous Fluids sputum body fluids Pus Homogenize the sample by vigorous vortexing for 5 10 min In the 15 ml falcon tube add the 2 NALC NaOH with the specimen sputum body fluids urine to an equal volume 1 1 and vortex for 1 min Incubate the mixture for 15 min at room temperature with occasional shaking Adjust the volume to 15 ml with sterile water or sterile phosphate buffer solution pH 6 8 and mix well Transfer 1 5 ml sample to a 2 ml Eppendorf tube using a pipette with a tip with aerosol barrier mark it and centrifuge at 10 000 g or 15 000 rpm for 10 min Discard the s
3. Mycobacterium avium subsp avium MTCC1723 The analytical sensitivity for NTM of the 3B MTB NTM KIT was determined by analyzing dilution series of known copy number of plasmid containing NTM target gene The 3B MTB NTM KIT tests demonstrated the ability to reproducibly detect the presence of NTM at the level of 50 copies ul 18 Page 3B BIOTUB QT Kit www 3bblackbio com MF 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio 5 1 and Biotools Labs S A Madrid Spain Table 2 Analytical sensitivity of the 3B MTB NTM KIT for NTM detection B Clinical Performance The clinical performance of the 3B MTB NTM KIT is evaluated regularly by analysing reference samples and diagnostic samples previously tested with a reference method mycobacteria culture 181 specimens derived from Sputum Aseptic fluids Broncho Alveolar Lavage Aseptic tissues and Pus collected in different laboratories and hospitals were tested for determining the diagnostic sensitivity and specificity of the 3B MTB NTM KIT Results were achieved by comparing the results obtained with the 3B MTB NTM KIT against results obtained by mycobacteria culture for the individual specimen 107 65 Table 3 Clinical performance of 3B MTB NTM KIT 19 Page BIOTUB QT Kit www 3bblackbio com MF yt 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L a
4. B amp M Labs S A Madrid Spain NTM DETECTION 1 REACTION PREPARATION a Prepare the PCR Mix as follows Multiplex Master Mix 10 ul MTB NTM Primer probe mix 2 ul Internal control Primer probe mix 2 ul b Mix well by inverting or quick vortexing and centrifuge briefly c Transfer 14 of the above prepared PCR Mastermix in 0 2 ml PCR tubes and close the tubes d For 14 of above reaction mix add 50 100 ng of DNA samples and make up the final volume 20 with nuclease free water DNA 50 100 ng Nuclease free Water Make up to 20 ul 2 PROGRAM SET UP Define the following setting for Temperature Profile and Dye Acquisition Step Temperature C Time Dye Acquisition Cycles 1 94 15 min 1 94 15 sec 2 60 30 sec 35 72 20 sec Yes 3 CHANNEL SELECTION Define the following setting for channel selection MTB specific DNA HEX None 43 NTM specific DNA NTM FAM None 50 Internal Control IC Tex Red None 50 15 Page 3B BIOTUB QT Kit www 3bblackbio com RESULT ANALYSIS 5 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio 5 1 and Biotools Labs S A Madrid Spain Mycobacterium tuberculosis MTB is Present present 1 Present Present Test sample is positive for MTB infection of MTB and Non tuberculosis Mycobacterium Present spe
5. add 1 ml sterile water to pellet Mix well and centrifuge at 15 000 rpm for 10 min Discard the supernatant and re suspend the pellet as per DNA extraction step Note if the pellet is not visible remove the supernatant leaving about 100 ul of the sample vii Broncho Alveolar Lavage BAL Homogenize the sample by vigorous vortexing for 5 10 min If sample is viscous dilute sample with water in 1 0 5 ratio and vortex to homogenize Centrifuge whole sample at 13 000 rpm for 15 min discard the supernatant If RBCs are present then give PBS wash to remove RBCs To pellet add 500 600 2 NALC NaOH solution and incubate for 15 20 min depending upon sample consistency at room temperature with intermittent vigorous vortexing If the sample is very viscous or appears to be tough thick then incubate it in water bath of temperature 40 50 C Neutralize the digested decontaminated specimens by adding Phosphate buffer saline PBS up to 15 ml and mix by inverting Centrifuge whole neutralized sample for 15 min at 13000 rpm Wash the pellet with 1ml PBS and centrifuged for 15 min at 13000 rpm Check the pH of supernatant and discard it If pH is neutral then wash pellet with 1ml water and if it is still alkali then give one more or two till it become neutral wash with PBS After neutralization give 1 more PBS washing Now add 1ml sterile water vortex pellet and centrifuge at 13000rpm for 10 min and carefully discard the supernatant
6. ml falcon tube To specimen add 296 NALC NaOH solution in 1 1 ratio Incubate for 15 20 min depending upon sample consistency at room temperature with intermittent vigorous vortexing If the sample is very viscous or appears to be tough thick then incubate it in water bath of temperature 40 50 C Neutralize the digested decontaminated specimens by adding Phosphate buffer saline PBS up to 15 ml and mix by inverting Centrifuge 1 5 ml neutralized sample for 15 min at 13000 rpm Wash the pellet with 1ml PBS and centrifuged for 15 min at 13000 rpm Check the pH of supernatant and discard it If pH is neutral then wash pellet with 1ml water and if it is still alkali then give one more or two till it become neutral wash with PBS After neutralization give 1 more PBS washing Now add 1ml sterile water to the pellet vortex and centrifuge at 13000 rpm for 10 min and carefully discard the supernatant Repeat this step once and proceed as per DNA Extraction step BIOTUB QT Kit www 3bblackbio com 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain iv Paraffin embedded tissue Add 1 ml of n Octane or Xylene to the tube containing several pieces of paraffin fragment Vortex vigorously and incubate at room temperature for about 30 min Vortex occasionally Centrifuge at 13 000 rpm for 3 min Pipette off supernatant If paraffin remains repeat s
7. with the kit before addition of Buffer BB3 and incubate at 37 C for 5 min Clogged columns Too much material used sample Do not use more sample material than recommended 25 mg for most tissue types If insoluble material like bones or hair remains in the lysate spin down the debris and transfer the clear supernatant to a new microcentrifuge tube before proceeding with addition of Buffer BB3 and ethanol Incomplete lysis Sample not thoroughly homogenized and mixed with Buffer BT1 Proteinase K The mixture has to be vortexed vigorously immediately after the addition of Buffer BT1 Decreased Proteinase K activity Store dissolved Proteinase K at 20 C for 6 months Reagents not applied properly Prepare Buffer BB3 Buffer BB5 and Proteinase K solution according to instructions Add ethanol to the lysates before loading them on the columns 22 Page 3B BIOTUB QT Kit www 3bblackbio com 5 3B BlackBio Biotech India Ltd A A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain All the components of 3B MTB NTM KIT should be stored at 20 C and stable until the date of expiry stated The reagents can be aliquoted and stored at 20 C in order to maintain the stability and sensitivity All the components of MTB DNA EXTRACTION KIT should be stored at room temperature and stable until the date of expiry stated Note Proteinase K
8. 3B BIOTUB QT Kit Real Time PCR Detection Z2 fil a gt l amp A Pi uu W m 2 CEAN De VLA lIoDallha Dz 39029024 Madrid S _ 4 740 nn 7 Iz 24 Pak T JIR JI OUD oT ANAIAL 1 E BlackBio Biotech A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain For detection and differentiation of Mycobacterium tuberculosis complex MTBC and nontuberculous mycobacteria NTM from human specimens For use with ABI Prism 7500 SDS 7500 Fast SDS ViiA 7 and QuantStudio 12K Flex Applied Biosystems Rotor Gene Q5 6 plex Platform Qiagen CFX384 Touch CFX96 Touch BioRad PicoReal 24 PicoReal 96 Thermo LightCycler 96 Roche LineGene K LineGene 9600 Bioer IN VITRO DIAGNOSTIC USE REF product No 111111 Was tests T perature limitation 2013 wal 3 BlackBio Biotech India Ltd 1 Page BIOTUB QT Kit www 3bblackbio com SF 92 3B BlackBio Biotech India Ltd
9. Repeat this step once and re suspend the pellet as per DNA Extraction step 11 Page 3B BIOTUB QT Kit www 3bblackbio com 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain C DNA Extraction Step Before starting DNA extraction protocol prepare the following Lysis Buffer BB3 Transfer the total contents of Buffer BB1 to Buffer BB2 and mix well Place the labels for Lysis Buffer BB3 on the bottle The resulting Lysis Buffer BB3 is stable for up to one year at room temperature Wash Buffer BB5 Add the below indicated volume of ethanol 96 100 to Wash Buffer BB5 Concentrate Mark the label of the bottle to indicate that ethanol was added Store Wash Buffer BB5 at room temperature 18 25 C for up to one year 48rxns 7 2 28 ml to each bottle 96 7 4 28 ml to each bottle 240 rxns 2x 40 ml 160 ml to each bottle Proteinase K Add the below indicated volume of Proteinase Buffer PB to dissolve lyophilized Proteinase K Proteinase K solution is stable at 20 C for up to 6 months 48 30mg 1 35 ml to each vial 96rxns 30mg 2 1 35 ml to each vial 240 rxns 75 mg 2 3 35 ml to each vial e Set on incubator or water bath to 50 C e Preheat Elution Buffer BBE to 70 C i Resuspension Resuspend the formed pellet in 0 2 1 Buffer BT1 depending on sampl
10. amination during handling Check for contamination of kit s component 2 No amplification signal with positive controls Incorrect PCR mixture Check whether all components are added Missing control sample during DNA mixing Be careful when pipetting Changing DNA during DNA mixing Write down sample number on the 1 5 ml micro centrifuge tube and the PCR tube Leaving reagents at room temperature for a long time or incorrect storage condition Please check the storage condition and the expiration date see the kit label of the reagents and use a new kit if necessary The PCR conditions do not comply with the protocol Repeat the PCR with corrected settings 3 Weak or no signal of the Reagent has been thawed and m Please mind the storage conditions frozen too often or exposed Internal Control in given in manual inappropriate storage conditions Red channel DNA of Poor quality may interfere with The PCR was inhibited the PCR reaction use a recommended isolation method 21 Page 3B BIOTUB QT Kit www 3bblackbio com 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain TROUBLESHOOTING DNA EXTRACTION 1 DNA yield poor Incomplete lysis Sample not thoroughly homogenized and mixed with Buffer BT1 Proteinase K The mixtur
11. cies NTM is present 2 Absent Present Test sample is negative for but positive for NTM Test sample is negative for MTB and 3 Absent Absent Present NTM 4 Absent Absent Absent PCR inhibition retest the sample Co infection but must be confirmed by further tests e g genotyping Detection of the Internal Control is not required for positive results in the FAM or HEX detection channels High MTB or NTM load in the sample can lead to reduced or absent Internal Control signals 16 Page 3B BIOTUB QT Kit www 3bblackbio com 592 BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio 5 1 Biotools Labs S A Madrid Spain Case 1 MTB Positive Case 2 NTM Positive Amplification Case 3 Negative 17 Page 3B BIOTUB QT Kit www 3bblackbio com sag 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio 5 1 and Biotools Labs S A Madrid Spain A Sensitivity 1 M tuberculosis The analytical sensitivity for M tuberculosis complex of the 3B MTB NTM KIT was determined by analyzing dilution series of known copy number of plasmid containing MTB target gene The 3B MTB NTM KIT test demonstrated the ability to reproducibly detect the presence of M tuberculosis at the level of 2 10 copies ul Table 1 Analytical sensitivity of the 3B MTB NTM KIT for MTB detection 2 NTM
12. e has to be vortexed vigorously immediately after the addition of Buffer BT1 Decreased Proteinase K activity Store dissolved Proteinase K at 20 C for 6 months Reagents not applied properly Prepare Buffer BB3 Buffer BB5 and Proteinase K solution according to instructions Add ethanol to the lysates before loading them onto the columns Suboptimal elution of DNA from the column Preheat Buffer BBE to 70 C before elution Apply Buffer BBE directly onto the center of the silica membrane Elution efficiencies decrease dramatically if elution is done with buffers with a pH lt 7 0 Use slightly alkaline elution buffers like Buffer BBE pH 8 5 Especially when expecting high yields from large amounts of material we recommend elution with 200 Buffer BBE and incubation of the closed columns in an incubator at 70 C for 5 min before centrifugation Poor DNA quality Incomplete lysis Sample not thoroughly homogenized and mixed with Buffer BT1 Proteinase K The mixture has to be vortexed vigorously immediately after the addition of Buffer BT1 Decreased Proteinase K activity Store dissolved Proteinase K at 20 C for 6 months Reagents not applied properly Prepare Buffer BB3 Buffer BB5 and Proteinase K solution according to instructions Add ethanol to the lysates before loading them on the columns RNA in sample If RNA free DNA is desired add 10 ul of RNase A solution 5 mg ml not supplied
13. e viscosity Transfer 200 pl of the resuspended sample to a new microcentrifuge tube not provided 12 Page 3B BIOTUB QT Kit www 3bblackbio com 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain ii Pre lyse sample Add 180 ul Buffer BT1 and 25 Proteinase solution Vortex to mix Be sure that the samples are completely covered with lysis solution Note f processing several samples Proteinase and Buffer BT1 may be premixed directly before use Do not mix Buffer BT1 and Proteinase K more than 10 15 min before addition to the sample Incubate at 56 C until complete lysis is obtained at least 1 h Vortex occasionally during incubation or use a shaking incubator Note Samples can be incubated overnight as well If RNA free DNA is crucial for downstream applications an RNase digest may be performed Add 20 ul RNase 20 mg ml solution not included and incubate for an additional 5 min at room temperature iii Lyse sample Vortex the samples Add 200 ul Buffer BB3 vortex vigorously and incubate at 70 C for 10 min Vortex briefly Note f insoluble particles are visible in Buffer BB3 centrifuge for 5 min at high speed e g 10 000 rpm and transfer the supernatant to a new micro centrifuge tube not provided iv Adjust DNA binding conditions Add 210 ul ethanol 96 100 to the sample and vortex vigo
14. ex MTC and Non tuberculosis mycobacteria NTM in clinical samples Mycobacterium tuberculosis Non tuberculosis DNA is extracted from samples amplified using Real Time Amplification and detected using fluorescent reporter dye probes specific for M tuberculosis complex and all mycobacterium genus In real time PCR the fluorescent signal is generated from the presence of an oligonucleotide probe specific for target DNA sequence The probe contains a fluorescent dye molecule on its 5 end and a quencher molecule on its 3 end The probe hybridizes with one of the chains of the amplified fragment During synthesis of a complementary chain Taq DNA polymerase which possesses 5 3 exonuclease activity cleaves the probe As a result the fluorescent dye and quencher dye are separated and the total fluorescence of reaction volume increases in direct proportion to the number of amplicon copies synthesized during PCR The fluorescent signal is measured in each cycle of reaction and the threshold cycle value is determined from the obtained curve The threshold cycle is proportional to the initial number of DNA copies in a sample and its value allows qualitative quantitative comparisons of analyzed and control samples In 3B MTB NTM detection kit is based on amplification of region upstream of the 65 kDa heat shock protein 65kDa hsp gene by primer and probes specific for M tuberculosis complex and mycobacterium genus In this kit there are t
15. hree independent reactions running in parallel in each tube the first detects Mycobacterium tuberculosis complex M tuberculosis M africanum M bovis M bovis BCG M microti HEX channel second detects all mycobacterium by genus specific probe FAM channel and the third detects internal control IC DNA Tex Red channel which allows excluding unreliable results 3 Page 3B BIOTUB QT Kit www 3bblackbio com REAGENTS RE 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain The Kit contains amplification reagents for performance of 48 96 amplification reactions Thaw and handle reagents on ice Do not freeze thaw Kit vials repeatedly In case of frequent use we recommend to aliquot the contents of the vials into 10 reactions each This will also rule out kit reagent contamination 3B MTB NTM REAL TIME KIT Reagent Description Volume in pL 48 reactions Volume 96 reactions Multiplex Master Hot start DNA polymerase Reaction Buffer dNTPs dATP dCTP dGTP 500 uL 500 uL X 2 ix dTTP MgCl and stabilizers Primer and probe mix for MTB NTM MTB and NTM detection 100 100 uL X 2 Primer probe mix e Primer probe and DNA mix Internal control for IC detection 100 uL 100 uL X 2 Primer probe mix Positive Control Positive Control 50 100 uL MTB Positi
16. nd Biotools B amp M Labs S A Madrid Spain C Clinical performance analysis 102 107 7 9 77 77 63 65 96 92 102 104 98 07 7 7 100 63 70 90 Table 4 Analysis of clinical performance D Specificity Specificity of 3B MTB NTM KIT is assured by selection of specific primers and probes as well as the selection of stringent reaction conditions The primers and probes were checked for possible homologies and to ensure that all relevant MTB and NTM are detected to all gene banks published sequences by sequence comparison analysis Specificity of 3B MTB NTM KIT was confirmed in laboratory clinical trials too It has been tested for cross reactivity with several common human pathogenic bacteria other than MTB NTM and as a result there was no case of real time PCR amplification was observed 20 Page 3B BIOTUB QT Kit www 3bblackbio com Reproducibility 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain The Reproducibility of specificity sensitivity and accuracy of 3B MTB NTM KIT were evaluated by reproducibility tests carried out at different points of time in the course of two months by different experimenters The results turned out to be the same confirming the reproducibility of the kit TROUBLESHOOTING REAL TIME PCR 1 Amplification signal in negative control Cross cont
17. nts in compliance with local authorities requirements e Samples should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices Infected material and disposable plasticware that was in contact with infected material must be treated with chlorine containing solutions e Clean and disinfect all sample or reagent spills using a disinfectant such as 0 5 96 sodium hypochlorite or other suitable disinfectant e Avoid contact with the skin eyes and mucosa If skin eyes and mucosa contact immediately flush with water seek medical attention 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 uni 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 24 Page 3B BIOTUB QT Kit www 3bblackbio com 5 3B BlackBio Biotech India Ltd AM A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain 1 ICMR Bulletin 2002 What is new in the diagnosis of Tuberculosis Part Techniques for the diagnosis of Tuberculosis Vol 32 No 8 2 Kapur V Li L L Hamrick MR Plikaytis BB Shinnick TM Telenti A et al 1995 Rapid Mycobacteri
18. rously v Bind DNA For each sample place one MTB DNA Spin Column into a Collection Tube Apply the sample to the column Centrifuge for 1 min at 10 000 rpm Discard the flow through and place the column back into the Collection Tube Note f the sample is not drawn completely through the matrix repeat the centrifugation step at 10 000 rpm Discard flowthrough 13 Page 3B BIOTUB QT Kit www 3bblackbio com 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain vi Wash silica membrane 1 wash Add 500 ul Buffer BBW Centrifuge for 1 min at 10 000 rpm Discard flow through and place the column back into the Collection Tube Add 600 ul Buffer BBS to the column and centrifuge for 1 min at 10 000 rpm Discard flow through and place the column back into the Collection Tube vii Dry silica membrane Centrifuge the column for 1 min at 10 000 rpm Residual ethanol is removed during this step viii Elute highly pure DNA Place the MTB DNA Spin Column into a 1 5 ml micro centrifuge tube not provided and add 100 pl pre warmed Buffer BBE 70 C Incubate at room temperature for 1 min Centrifuge 1 min at 10 000 rpm xi 50 100 ng of the eluted DNA should be used for the amplification 14 Page 3B BIOTUB QT Kit www 3bblackbio com 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools
19. should be stored at 20 C once dissolved e Adjustable pipettes with sterile filter or positive displacement tips 2 NALC NaOH e Phosphate Buffer Saline PBS Disposable powder free gloves e Sterile bidistilled water Sterile 1 5 ml and 2 ml microcentrifuge tubes 50 conical tubes e Vortex mixer e Heating block for incubation at 70 C e Water Bath e 3BTermi DNA Tor or equivalent in order to remove DNA from working surfaces e Desktop centrifuge e Real time PCR e Laminar airflow cabinet e vials 0 2 ml thin walled e 96 100 ethanol Personal protection equipment lab coat gloves goggles 23 Page 3B BIOTUB QT Kit www 3bblackbio com 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain GENERAL PRECAUTIONS The user should always pay attention to the following e Use sterile pipette tips with aerosol barriers and use new tip for every procedure Thaw all components thoroughly at room temperature before starting detection e When thawed mix the components and centrifuge briefly e Use disposable gloves laboratory coats and protect eyes while samples and reagents handling Thoroughly wash hands afterwards 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 samples and unused reage
20. tep 1 and 2 Add 1 ml of ethanol 96 10096 and then vortex it Centrifuge for 3 min at 13 000 rpm Pipette out supernatant Repeat step 3 and 4 Pipette off as much of the ethanol as possible Incubate the open tube at 37 C until the ethanol has evaporated 15 min Discard the supernatant and re suspend the pellet as per DNA extraction step Note if the pellet is not visible remove the supernatant leaving about 100 of the v Stool sample Homogenize the sample by vigorous vortexing for 5 10 min Mix the stool at least 1 with 10 ml of 2 NALC NaOH vortex vigorously for 30 sec and incubate for 15 min at room temperature Centrifuge for 10 min at 1 500 rpm transfer 1 5 ml of the supernatant to 2 ml Eppendorf tube using a pipette with a tip with aerosol barrier mark it and centrifuge at 10 000 g or 15 000 rpm for 10 min Discard the supernatant and re suspend the pellet as per DNA extraction step Note if the pellet is not visible remove the supernatant leaving about 100 of the sample vi Blood Bone marrow aspiration Menstrual blood Take 0 5 ml of whole blood or Bone marrow aspiration and mix well with 1 ml of sterile water 10 Page 3B QT Kit www 3bblackbio com 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain Centrifuge for 10 min at 10 000 g or 15 000 rpm Discard the supernatant and
21. um species assignment and unambiguous identification of mutations associated with antimicrobial resistance in Mycobacterium tuberculosis by automated DNA sequencing Arch Pathol Lab Med 119 131 8 3 Kim H Kim SH Shim TS Kim MN Bai GH et al 2005 Differentiation of Mycobacterium species by analysis of the heat shock protein 65 gene hsp65 Int J Syst Evol Microbiol 55 1649 1656 4 Tobler N E M Pfunder K Herzog J E Frey and M Altwegg 2006 Rapid detection and species identification of Mycobacterium spp using real time PCR and DNA microarray J Microbiol Methods 66 116 124 The user should always pay attention to the following This test is for use with Sputum Aseptic fluids body fluid blood bone marrow aspiration menstrual blood Bronchial washing Aseptic tissues fresh tissues paraffin embedded tissue endometrium tissue Pus Urine and Stool Store DNA samples at 20 C until ready for use and keep on ice during use Avoid microbial contamination of reagents when removing aliquots from reagent tubes The use of sterile disposable pipette tips is recommended Specimens should be handled as if infectious using safe laboratory procedures Thoroughly clean and disinfect all work surfaces with 0 596 Sodium Hypochlorite in de ionized or distilled water 25 Page 3B BIOTUB QT Kit www 3bblackbio com
22. upernatant add 1 ml of PBS solution and mix well Note if the pellet is not visible remove the supernatant leaving about 100 of the sample Centrifuge at 10 000 g or 15 000 rpm for 10 min discard the supernatant and re suspend the pellet as per DNA extraction step ii Non viscous Fluids aspiration body fluids urine pleural fluid 8 Page Homogenize the sample by vigorous vortexing for 5 10 min Transfer 1 5 ml of the sample to a 2 ml Eppendorf tube using a pipette with a tip with aerosol barrier mark it and centrifuge at 10 000 g or 15 000 rpm for 10 min Discard the supernatant and re suspend the pellet as per DNA extraction step 3B BIOTUB QT Kit www 3bblackbio com 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain Note if the pellet is not visible remove the supernatant leaving about 100 ul of the sample iii Fresh tissue Biopsy Endometrium tissue 9 Page If RBCs are present then give PBS wash to remove RBC s totally If tissues are soft then add NALC NaOH solution and start processing as described below If tissues are tough then grind and homogenize it with PBS in sterile mortar pestle and process as described below Process sample in original sample container If sample is in syringe or small container then transfer and process the sample in 50 ml Falcon tube preferred for more surface area or 15
23. ve Control Positive Control 50 100 uL NTM Negative Control e Sterilized water 50 uL 100 uL 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain MTB DNA EXTRACTION KIT MTB DNA Extraction Kit 48 Reactions 96 Reactions 240 Reactions Reagent Volume Volume Volume Buffer BT1 20 ml 20 ml X 2 100 ml Buffer BB1 12 12 2 60 ml Reagent BB2 3 ml 3mlX2 15 ml Buffer BB5 Concentrate 7mlX2 7 4 40 mI X 2 Buffer BBW 30 ml 30mlX2 75mlx2 Buffer BBE 15 ml 15mlX2 75 ml Proteinase K Lyophilized 30 mg 30mgX2 75 mg x2 Proteinase Buffer 1 8 ml 1 8 2 8 Spin Columns 50 100 250 2 ml Collection Tubes 100 200 500 Label for Buffer BB3 1 1 1 User Manual 1 1 1 3B BlackBio Biotech India Ltd A joint venture of Kilpest India Limited 2B BlackBio S L and Biotools B amp M Labs S A Madrid Spain INSTRUCTIONS FOR USE A Sample collection Sputum samples Tissues Collection of early morning sputum specimen is optimal Rinse the mouth with water and then should cough deeply to expectorate sputum directly into the sterile leak proof container Samples should be stored at temperatures of 2 8 C or freezing Fresh biopsies of up to 5 mm must be used Any tissue must be collected aseptically into a sterile container without fixati
24. ves or preservatives To keep moist add sterile saline to the dried specimen Keep refrigerated until transport Biopsy should be stored at 15 8 C Paraffin embedded tissues can also be used in cases where the tissue fixation method does not degrade DNA and purification of DNA is performed with methods specific for this kind of sample An early morning midstream specimen should be collected Multiple specimens over several days are optimal to obtain a positive specimen Samples should be stored at temperatures of 2 8 C or freezing Aseptic fluids body fluid bone marrow blood Bronchial washing 6 Page Body fluids spinal pleural pericardial synovial ascitic bone marrow bronchial secretions washings should be aseptically collected in a sterile container using aspiration techniques or surgical procedures BIOTUB QT Kit www 3bblackbio com MF 3B BlackBio Biotech India Ltd A joint venture Kilpest India Limited 2B BlackBio 5 1 and Biotools Labs S A Madrid Spain e For fluids that may clot sterile potassium oxalate 0 01 0 02 ml of 10 neutral oxalate per ml fluid or heparin 0 2 mg per ml should be added Stool should be passed into a clean and dry container e Carefully open the specimen vial and collect a small sample with the help of a sterile spoon in to a new sterile vial e Close the vial tightly and shake to mix well
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