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1. FAM Quencher None to test TB DNA 2 Select HEX or VIC channel Reporter HEX VIC Quencher None to test TB internal control 3 Set passive reference none SET SAMPLE VOLUME 50 3 2 2 For Roche Light Cycler 480 Choose New Experiment Click Dual Color Hydrolysis Probe UPL Probe in the drop down menu of setup panel Do the following in the drop down menu of Customize 1 Select FAM channel to test TB DNA 2 Select VIC HEX Yellow 555 channel to test internal control 3 Set reaction volume 50 Set cycle parameters the time parameter varies according to instruments ABI STRATAGENE SERIES Step Temperature UNG enzyme reaction 50 C Taq Enzyme activation 94 C Denaturation 94 C Annealing Extension Fluorescence collection Device cooling optional NOTE Due to the device ABI 7500 s technical specification it can t be set at 30 seconds but 31 seconds When the setting is completed save settings and carry out the reaction procedure RESULT ANALYSIS Refer to user s manual for each instrument to make the setting When the reactions are completed results will be saved automatically After analysis adjust Start End and Threshold values of Baseline of the graph users can adjust according to the actual situation Start value can be set between3 15 end value between5 20 Adjust the amplification curve of negative control to be flat or below threshold Click Analyze2. processing delivery and storage quality Any deviation from the stated procedure will lead to an inaccurate detection result Cross contamination during specimen processing may also result in a false positive result PRODUCT PERFORMANCE INDEX When the kit is used to detect the enterprise s work references the consistency rate for both negative and positive reaches 100 Precision test shows excellent reproducibility in both intra batch and inter batches with its coefficient of variation of Ct value lt 10 and its coefficient of variation of concentration lt 50 The sensitivity of this kit is 1 bacterium ml It shows no cross reaction with Mycobacterium Avium Land Mycobacterium Amur Mycobacterium Mycobacterium Kansasii Asian Mycobacterium M scrofulaceum Mycobacterium Gordon Mycobacterium Turtle pus Mycobacterium Accidental Mycobacterium Mycobacterium phlei Brazilian Nocardia Corynebacterium pekinense Pneumococcus Legionella pneumophilia Bordetella pertussis MP EBV and respiratory syncytial virus PRECAUTIONS 1 The product can only be used for in vitro diagnosis Please read carefully the product manual before operation 2 Please familiarize with and learn the operation procedures and precautions for each instrument before performing tests Conduct quality control for each test 3 Laboratory management shall strictly follow management practices of PCR gene amplification laboratory test staff must receive professional t
3. Mycobacterium tuberculosis DNA FLUORESCENCE DIAGNOSTIC KIT PCR FLUORESCENCE PROBING For Professional Use Read the pack Insert before use provided along with the kit REF Mtdf 48 INTENDED USE Mycobacterium tuberculosis TB is a pathogenic bacterium that causes tuberculosis It is likely to infect all human tissues and organs especially the lungs Early diagnosis and treatment are important for effective control of TB In recent years with the development of Molecular Biology nucleic acid fluorescence quantitative PCR method based on the mycobacterium tuberculosis nucleic acid has drawn more and more attention from researchers This diagnostic kit is an in vitro nucleic acid amplification test for the detection of Mycobacterium tuberculosis TB DNA in human sputum It is intended for use as an aid in diagnosing a TB infection TEST PRINCIPLE The diagnostic kit uses a nucleic acid lysis buffer to allow rapid lysis and release of TB DNA from a sputum specimen By applying real time fluorescence quantitative PCR technology this test utilizes a pair of specific primers which are designed to target at a conserved sequence of TB DNA a specific fluorescence probe accompanied with PCR mix to achieve fast detection of TB DNA through fluorescent signal changes The PCR detection system uses UNG enzyme dUTP contamination proof system which can fully degrade possible unwanted side products to avoid a false positive result PACK SIZE 48Tests K
4. imens performed at specimen processing region 2 1 Processing specimen Add into specimen 4 NaOH solution whose volume equals 2 3 times of specimen Vortex it and hold it for 30 min to allow it liquefy Take 500uI liquefied specimen to a 1 5 ml centrifuge tube avoid aspirating apparent solid impurities Centrifuge it at 12000 rpm for 3 minute Aspirate and discard the supernatant Add 1 ml sterile saline and centrifuge it at 12000 rpm for 3 minute Aspirate and discard the supernatant Add into 50ul nucleic acid lysis buffer Treat it at 100 C for 10 min Centrifuge it at 12000 rpm for 3 min Take the supernatant as specimen for later use 2 2 Adding specimens negative controls and specimens are processed in synchronization 2 2 1 Add Sul of specimen Negative control Positive control and Quantitative references respectively into each PCR reaction tube Page 2 of 5 2 2 2 Add 40ul of PCR mix into each tube Remove the bubbles and cover the tube Centrifuge it at 2000 rpm for 30 seconds 3 PCR Amplification performed at amplification and analysis region refer to user s manual for each instrument to do the settings 3 1 Place the PCR reaction tube into the specimen well of the amplification device Set negative control positive control quantitative references A D and unknown specimens in corresponding sequence and set specimen names 3 2 Select PCR test channel 3 2 1 For ABI Stratagene series 1 Select FAM channel Reporter
5. it COMPONENTS OF THE KIT S Specification amp Reagent name quantity DNA lysis buffer 2 5ml Tube x1 Tube KCI SDS Surfactin Main ingredients Enzyme mixture 96ul Tube x1 Tube DNA Polymerase Uracil DNA g ycolase TB PCR Mix 912ul Tube x 2 Tube Primer Probe dNTPs Mg PCR Buffer Quantitative TB Positive specimen Inactivated Quantitative TB Positive specimen Inactivated Quantitative TB Positive specimen Inactivated Quantitative TB Positive specimen Inactivated TB Quantitative Reference A 50ul Tube x1 Tube TB Quantitative Reference B 50ul Tube x1 Tube TB Quantitative Reference C 50ul Tube x1 Tube TB Quantitative Reference D 50ul Tube x1 Tube TB Negative Control 50ul Tube x1 Tube Sterile saline Quantitative TB Positive specimen Inactivated Positive internal reference cloning plasmid without TB target sequence TB Positive control 50ul Tube x1 Tube TB Internal Control 50ul Tube x1 Tube Page 1of5 NOTE 1 Do NOT mix components from different lots 2 All biological specimens in the detection kit should be handled as if infectious though they have been inactivated 3 Self prepared reagent sterile saline and 4 NaOH solution STORAGE CONDITION AND TERM OF VALIDITY The detection kit should be stored in sealed pouch at 20 5 C protected from light The term of validity is 12 months Care should be taken to av
6. oid re freezing and re thawing APPLICABLE INSTRUMENT The diagnostic kit is applicable to fluorescence PCR instruments such as ABI7500 and Mx3000P SPECIMEN REQUIREMENTS 1 Applicable specimen type Human Sputum 2 Collection of specimen It is recommended to collect the first sputum in the morning First rinse mouth with water Make a hard cough and collect the sputum in the deep and keep it in a sterile collection tube Seal it and send it for detection 3 Storage and delivery of specimens Specimens collected via the above mentioned method can be used for immediate detection or stored at 2 8 C for not more than 24 hours or below 20 C for a longer term of storage Care should be taken to avoid re freezing and re thawing Specimens should be transported in a sealed frozen pitcher with ice or in a sealed foam box with ice TEST METHOD 1 Preparation of reagent Performed at reagent preparation region 1 1 Take out each component from the detection kit and place them at room temperature When the components temperature has reached room temperature mix them for later use 1 2 Refer to quantities of test specimens negative controls and positive controls and quantitative references A D pipette appropriate quantities of PCR mix enzyme mixture and internal control PCR mix 38 ul test enzyme mixture 2 I test internal control 1p test fully mix them and centrifuge it instantaneously for later use 2 Processing and adding spec
7. raining testing process must be performed in separated regions all used consumables should be of sterile single use specific instruments and devices should be used for each stage All lab devices used in different stages and regions should not be cross used Page 4of5 R O 10 2014 4 All specimens for detection should be handled as if infectious Wear protective disposable gloves laboratory coats and eye protection when handling specimens and kit reagents Wash hands thoroughly after handling specimens and test reagents Handling of waste must meet relevant requirements outlined in Common Criteria of Bio safety for Microbiology Biomedical Laboratory and Medical Wastes Management Regulations by Health Department 5 Before use all reagents must be fully thawed at room temperature and mixed thoroughly BIBLIOGRAPHIES 1 Benjamin A Pinsky Niaz Banaei Multiplex Real Time PCR Assay for Rapid Identification of Mycobacterium tuberculosis Complex Members to the Species Level Journal of Clinical Microbiology 2008 46 2241 2246 2 Marion Blaschitz1 Dzenita Hasanacevici PeterHufnagl et al Real time PCR for single nucleotide polymorphism detection in the 16S rRNA gene as an indicator for extensive drug resistance in Mycobacterium tuberculosis J Antimicrob Chemother 2010 45 1093 1110 3 van Doorn HR An DD de Jong MD Lan NT et al Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe
8. real time PCR Int J Tuberc Lung Dis 2008 7 736 42 4 B J Renton P D Morrell Direct real time PCR examination for Mycobacterium tuberculosis in respiratory samples can be cost effective Health 200 9 11 63 66 aad Manufactured in India by BHAT BIO TECH INDIA P LTD 11 A 4TH CROSS VEEREASANDRA INDUSTRIAL AREA ELECTRONICS CITY BANGALORE 560100 KARNATAKA INDIA TEL 080 3319 4000 30 LINES FAX 080 3319 4001 www bhatbiotech com Page 5of5
9. to implement the analysis and make sure each parameter satisfies the requirements given in Quality Control Go to Plate window to record the detected Ct value Page 3 of 5 QUALITY CONTROL 5 1 TB Negative Control no display of Ct value but the internal control is detected as positive Ct value lt 40 5 2 TB Positive Control detected Ct value lt 30 5 3 Four TB Quantitative References all are tested as positive 5 4 The above requirements must be satisfied at the same time in the same test otherwise the test is treated as invalid and needs to be re tested REFERENCE RANGE Through the research on reference values the Ct reference value of target gene is 39 and the Ct reference value of internal control is determined to be 40 EXPLANATION OF DETECTION RESULT Determination of Negative and Positive results 1 Specimens which are detected with a Ct value lt 39 are reported as TB DNA positive 2 For specimens which are detected with a Ct value gt 39 and internal control is detected as positive Ct value lt 40 report that TB DNA is lower than detection limit If the Ct value of internal control gt 40 or there is no display of Ct value the test result is invalid An investigation should be conducted to find out reasons and retest it If retests still show invalid result please contact Bhat Bio Tech India Pvt Ltd Bangalore LIMITS OF DETECTION METHOD Detection result of specimen is related to specimen collection
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