Chip-based Real Time PCR test for Mycobacterium tuberculosis
Contents
1. of this test requires appropriate specimen collection handling storage and transportto the testsite 2 Though very rare mutations within the highly conserved regions of the target genome where the Truenat assay primers and or probe bind may result in the under quantitation of or a failure to detectthe presence of the concerned pathogen 3 The instruments and assay procedures are designed to minimize the risk of contamination by PCR amplification products However it is essential to follow good laboratory practices and ensure careful adherence to the procedures specified in this package insert for avoiding nucleic acid contamination from previous amplifications positive controls orspecimens 4 A specimen for which the Truenat assay reports Not Detected cannot be concluded to be negative for the concerned pathogen As with any diagnostic test results from the Truenat assay should be interpreted in the context of other clinical and laboratory findings Spills of potentially infectious material should be cleaned up immediately with absorbent paper tissue and the contaminated area should be decontaminated with disinfectants such as 0 5 freshly prepared sodium hypochlorite 10 times dilution of 5 sodium hypochlorite household bleach before continuing work 2 Sodium hypochlorite should not be used on an acid containing spill unless the spill area is wiped dry first Materials used to clean spills including gloves should b2. AG Sample Prep device and Trueprep MAG Sputum Sample Prep Kit Six 6 uL of the purified DNA is dispensed into the reaction well of the Truenat MTB chip based Real Time PCR test The Truenat MTB chip based Real Time PCR test is then inserted into the Truelab Uno Real Time micro PCR Analyzer where thermal cycling takes place A positive amplification causes the dual labeled fluorescent probe in the Truenat MTB chip based Real Time PCR test to release the fluorophore in an exponential manner which is then captured by the built in opto electronic sensor and displayed as amplification curve on the analyzer screen ona real time basis during the test run The Cycle threshold Ct is defined as the number of amplification cycles required for the fluorescent signal to cross the threshold i e exceed the background signal Ct levels are inversely proportional to the amount of target nucleic acid in the sample i e the lower the Ct evel the greater is the amount of target nucleic acid in the sample Ct value is linearly correlated with he initial load of target DNA present in the sample enabling quantitative estimation of the analyte Standard values for every batch are preset in the Truenat MTB chip based Real Time PCR testand he analyzer automatically compares these with the Ctvalue of the testsample to provide a quantitative result Inthe case of negative samples amplification does not occur anda horizontal amplification curve is displayed
3. Hospital and Medical Research Centre Nikam et al in press PLOS ONE 226 sputum specimens from suspected TB patients were analyzed using smear microscopy culture in house nested PCR and Truenat MTB Pelleted sputum specimens were re suspended in lysis buffer from the Trueprep MAG Sputum kit and processed using the Trueprep MAG Sample Prep Device followed by PCR on Truenat MTB chip based Real Time PCR test Results were compared with a Composite Reference Standard CRS comprising microbiological tests clinical and radiological findings and patient history The results are tabulated below Smear Culture In house Nested PCR Truenat MTB e ve e Ve e ve e ve CRS ve 120 71 141 50 173 18 174 17 CRS ve 00 35 00 35 03 32 00 35 Sensitivity 62 83 73 82 90 58 91 10 Specificity 100 100 91 43 100 PPV 100 100 98 30 100 NPV 33 02 41 18 64 00 67 31 CRS Composite Reference Standard PPV Positive Predictive Value NPV Negative Predictive Value The results show that Truenat MTB was the most sensitive 91 10 and specific 100 test compared with the Composite Reference Standard Truenat MTB also showed high sensitivities of 99 12 among smear positive and culture positive specimen and 75 86 among smear negative and culture positive specimen Another study evaluating the Truenat MTB test was performed using a characterized 100 sample panel from suspected TB patients re
4. before disposal as per the standard medical waste disposal guidelines Disinfect the solutions and or solid waste containing biological samples before discarding them according to local regulations Samples and reagents of human and animal origin as well as contaminated materials disposables neutralized acids and other waste materials must be discarded according to local regulations after decontamination by immersion in a freshly prepared 0 5 of sodium hypochlorite for 30 minutes 1 volume of 5 sodium hypochlorite for 10 volumes of contaminated fluid or water Do notautoclave materials orsolutions containing sodium hypochlorite Chemicals should be handled in accordance with Good Laboratory Practice and disposed off according to the local regulations w N or SPECIFIC PERFORMANCE CHARACTERISTICS ASSAY RANGE AND LIMIT OF DETECTION To determine the efficiency of the PCR master mix and primer probe set for MTB DNA amplification standard curves were developed using serial log dilutions of target DNA Plasmids carrying cloned MTB PCR amplicons were used as template DNA The plasmids were quantified by UV spectrophotometry before dilutions to determine the colony forming units per milliliter cfu ml The Truenat MTB assay is linear over 7 log MTB DNA dilutions and can detectas low as 400 cfu ml of sputum ANALYTICAL INCLUSIVITY USING WHO TDR Strain Bank Sensitivity All 235 strains in the TDR TB Strain Bank were teste
5. c Truenat MTB Chip based Real Time PCR test for Mycobacterium tuberculosis INTENDED USE Truenat MTB REF 601030005 601030020 is a chip based Real Time Polymerase Chain Reaction PCR test for the quantitative detection and diagnosis of Mycobacterium tuberculosis MTB in human sputum specimen and aids in the diagnosis of infection with MTB Truenat MTB runs on the Truelab Uno Real Time micro PCR Analyzer INTRODUCTION Tuberculosis TB is an infectious disease caused predominantly by the bacillus Mycobacterium tuberculosis It typically affects the lungs pulmonary TB but can affect other sites as well extra pulmonary TB Tuberculosis TB is the second largest killer worldwide after HIV and is the leading cause of death in HIV patients Pulmonary TB spreads through air and is highly contagious Over 80 of TB infections are pulmonary and if left untreated a pulmonary TB patient can infect up to 10 15 other people through close contact over the course of a year Due to the highly infectious nature of pulmonary TB itis importantto diagnose and treat the disease very early Despite the availability of highly effective treatment for decades TB remains a major global health problem mainly because of poor case detection The most common method for diagnosing pulmonary TB worldwide is sputum smear microscopy However sensitivity of direct smear microscopy is low and estimates range from 30 to 70 Itis even lower in case of HIV infe
6. cted patients Culture is more sensitive than microscopy and is considered the current gold standard Culture requires specialized and controlled laboratory facility and highly skilled manpower and takes 3 to 6 weeks to provide the result Molecular techniques such as polymerase chain reaction PCR or Real Time PCR are much more sensitive than microscopy and culture However PCR or Real Time PCR tests have so far been restricted to centralized reference laboratories as they require skilled manpower and elaborate infrastructure Also the turnaround time for results could take a few days The Truelab Real Time micro PCR System enables decentralization and near patient diagnosis of MTB by making real time PCR technology rapid simple robust and user friendly and offering sample to result capability even at resource limited settings This is achieved hrough a combination of lightweight portable mains battery operated Truelab Uno Real Time micro PCR Analyzer and Trueprep MAG Sample Prep Device and room temperature stable Truenat chip based Real Time PCR test and Trueprep MAG Sample Prep kits so that even the peripheral laboratories with minimal infrastructure and minimally trained technicians can easily perform hese tests routinely in their facilities and report PCR results in less than an hour Moreover with these devices PCR testing can also be initiated in the field level onsite Truenat MTB is a disposable room temperature stabl
7. d in a blinded study to check the sensitivity of Truenat MTB againsta panel ofdiverse MTB strains Specimen used Providedby MTB Positive MTB Negative Truenat MTB result 235 strains of heat ITM Antwerp 233 02 Identified all inactivated Mycobacterial samples correctly cellsuspension Truenat MTB identified all 233 M tuberculosis strains and obtained a negative result for the 2 non uberculous mycobacteria that were included M terrae and M avium in the test panel Truenat MTB correctly identified MTB DNA in the TDR TB Strain Bank panel ANALYTICAL EXCLUSIVITY Primer Specificity Various strains of Non tuberculous Mycobacteria NTM including Mycobacterium fortuitum Mycobacterium abscessus Mycobacterium kansasii I Mycobacterium avium Mycobacterium gordanae Mycobacterium smegmatis and Mycobacterium szulgai were spiked into negative sputum sample and tested using Truenat MTB chip based Real Time PCR test Cultures of following common bacterial isolates Klebsiella pneumoniae Streptococcus sp Acinetobacter sp Escherichia coli Salmonella typhi Enterococcus sp and Pseudomonas aeruginosa were also tested using the Truenat MTB chip based RealTime PCR test The NTM panel and the bacterial isolates were not detected by the Truenat MTB chip based Real Time PCR test indicating its specificity for the MTB complex CLINICAL VALIDATION A pilot study was conducted at P D Hinduja National
8. e chip based Real Time PCR test with dried down PCR reagents for performing Real Time PCR test for detection of Mycobacterium tuberculosis and runs on the Truelab Real Time micro PCR Analyzer Itrequires only six 6 uL of purified DNA to be added to the reaction well for the analysis The intelligent chip also carries test and batch related information including standard values for quantitation The Truenat MTB chip based Real Time PCR testalso stores information of used test to prevent any accidental re use of the test NOTE Truelab Truelab Uno Trueprep MAG Truepet Truenat are all registered trademarks of Molbio Diagnostics P Limited The Truelab Real Time micro PCR Analyzer is protected by the following patents and patents pending IN 2313 CHE 2007 WO 2009 047804 and corresponding claims of any foreign counterpart s thereof The Truenat micro PCR chip is protected by the following patents and patents pending IN 2312 CHE 2007 WO 2009 047805 and corresponding claims of any foreign counterpart s thereof The Truenat MTB chip based Real Time PCR testis protected by the following patents and patents pending IN 796 CHE 2012 and corresponding claims of any foreign counterpart s thereof PRINCIPLE OF THE TEST Truenat MTB works on the principle of Real Time Polymerase Chain Reaction The DNA from the patient sputum sample is first extracted using Trueprep M
9. e disposed off as potentially bio hazardous waste e g ina biohazard waste container TEST PROCEDURE Please also refer Section 4 in the Truelab Uno Real Time micro PCR Analyzer user manual Switch on the Truelab Uno Real Time micro PCR Analyzer touch screen Selectuser and enter password Selectthe test profile for MTB on the Analyzer screen Enter the patient details as prompted in the Truelab Uno Real Time micro PCR Analyzer screen Press StartReaction Press the eject button to open the chip tray Open a pouch of Truenat MTB and retrieve the chip based RealTime PCR test Label the chip with the patient ID using a marker pen at the space provided on the back side of the chip 9 Place the Truenat MTB chip based Real Time PCR test on the chip tray without touching the white reaction well The reaction well should be facing up and away from the Analyzer Gently press the chip to ensure thatitis seated in the chip tray properly 10 Using the filter barrier tip provided in the pouch pipette six 6 uL ofthe purified DNA from the E lute Collection Tube into the centre of the white reaction well Take care notto scratch the internal well Pew Coo QUALITY CONTROL PROCEDURES surface and notto spill elute on the outside of the well 11 Slide the chip tray containing the Truenat MTB chip based Real Time PCR test loaded with the sample into the Truelab Uno Real Time micro PCR Analyz
10. er 12 Press the power button on the Analyzer to turn iton The green LED should glow 13 Press Doneon the Please Load Sample Alert message 14 Atthe end of the test observe the optical plot for any irregularities Refer to the Truelab Uno RealTime micro PCR Analyzer manual 15 Read the resultfrom the screen 16 Press the power button on the Analyzer to turn it off The green LED should stop glowing 17 Take outthe Truenat MTB micro PCR chip atend of the test and dispose it off as per the section on Disposal and Destruction Section 16 18 Turn on Truelab micro PCR printer and select print on the screen for printing out hard copy of the results Test results are automatically stored and can be retrieved any time later R efer to Truelab Uno Real Time micro PCR Analyzer manual 19 Switch off the Truelab Uno Real Time micro PCR Analyzer touchscreen RESULTS amp INTERPRETATIONS Two amplification curves are displayed on the Truelab Uno Real Time micro PCR Analyzer screen when optical plotis selected to indicate the progress of the test Both the target and the internal positive control IP C curves will take a steep exponential path when the fluorescence crosses the threshold value in case of positive samples The Ctwill depend on the number of bacterial genomes in the sample The target curve will remain horizontal throughout the test duration and the IPC curve will take an exponential path
11. ferred to a hospital in South East Asia The study involved processing of 500u ofeach sputum specimen using the Trueprep MAG Sputum Sample Prep Kiton the Trueprep MAG Sample Prep Device The purified nucleic acids were tested using Truenat MTB chip based Real Time PCR test and MTB specific primers and probe on a commercial real time PCR machine Sample Type Commercial real time PCR machine result Truenat MTB S C 40 40 100 40 40 100 S C 30 40 75 30 40 75 S C 0 20 nil detected 0 20 nil detected The Truenat MTB chip based Real Time PCR test was able to detect 100 of the S C samples 40 40 75 of S C samples 30 40 and gave a negative result for 100 of the S C samples 20 20 REPRODUCIBILITY AND PRECISION Assay reproducibility and precision was determined across different users analyzers and reagent lots Serial log dilutions of MTB genomic DNA were used for this purpose The same sample panel was provided to 3 users who ran iton 3 differentTruelab analyzers with 3 different lots of Truenat MTB chip based Real Time PCR test High inter user inter analyzer and inter lot reproducibility was observed avg standard deviation 0 3 Ct and no significant difference was observed in the Ct values obtained from different users analyzers or lots INTERFERING SUBSTANCES In order to study if there is any inhibition in the amplification due to potential interfering substance
12. gbaini Emovon 2009 Current Trends In The Laboratory Diagnosis of Tuberculosis Benin ournal of P ostG raduate Medicine Vol 11 Supplemental pp 79 90 7 Dye C Watt C Bleed D M et al 2005 Evolution of tuberculosis control and prospects for reducing tuberculosis incidence prevalence and deaths globally AMA 2005 293 2767 2775 8 Kennedy et al 1994 Polymerase Chain Reaction for Assessing Treatment Response in Patients with Pulmonary Tuberculosis The J ournal of Infectious Diseases Vol 170 No 3 pp 713 716 9 ikam et al in press PLOS ONE SYMBOLKEYS BE Refer package insert for instructions n vitro Diagnostic Test o Store For 30c Catalogue Notfor medicinal use 2 30 C Number use only ual Manufacturer Date of Expiry Batch Number Lot Number Date of Manufacture before use z Contains sufficient 7 for lt n gt tests ul Manufactured by VAAS molbio Molbio Diagnostics Pvt Ltd M 46 47 Phase Ill B Verna Industrial Estate Verna Goa 403 722 India E mail sales tulipgroup com TNMTB 0513 VER O2
13. in case of negative samples In case the IPC curve remains horizontal in a negative sample the test is considered as Invalid At the end of the test run the results screen will display DETECTED for P ositive result or NOT DETECTED for Negative result The resultscreen would also display the Ct value and the colony forming units per milliliter cfu ml for positive specimen The result screen also displays the validity of the test run as VALID or INVALID Invalid samples have to be repeated with fresh specimen from the sample preparation stage Note IPC will co amplify in most positive cases also in some specimen having a high target load the IPC may not amplify however the testrunis still considered valid To ensure that the Truelab Uno Real Time micro PCR Analyzer is working accurately run positive and negative controls from time to time The Truenat Universal Control Kit containing Positive Control and Negative Control must be ordered separately It is advisable to run controls under the following circumstances Whenever a new shipment of test kits is received When opening a new testkitlot If the temperature of the storage area falls outside of 2 30 C By each new user prior to performing testing onclinicalspecimen DISPOSALAND DESTRUCTION 1 Submerge the used Truenat MTB chip based RealTime PCR testin freshly prepared 1 sodium hypochlorite solution for 20 minutes
14. me PCR testfor Mycobacterium tuberculosis 2 DNase amp R Nase free pipette tip 3 Desiccantpouch B Package Insert 601030005 VW 5T STORAGE AND STABILITY Truenat MTB chip based Real Time PCR testis stable for one year from the date of manufacture if stored between 2 30 C Itis also stable for upto three 3 months at temperatures up to 40 C Avoid exposure to light 601030020 20T MATERIALS REQUIRED BUT NOTPROVIDED WITHTHE KIT Truelab Real Time micro PCR Workstation R EF 603010001 consisting of 1 Trueprep MAG Sample Prep Device REF 603040001 2 Truelab Uno Real Time micro PCR Analyzer REF 603020001 3 Truelab micro PCR Printer REF 603050001 4 Truepet Precision Micropipettes 6uL 50uL 100uL 500uL 1000uL REF 604010006 604020050 604030100 604040500 604051000 5 Marker pen Also required additionally are Trueprep MAG Sputum Sample Prep Kit REF 602020050 Truenat Universal Control Kit REF 601100008 DNase and RNase free pipette tips 2 200uL 200 1000uL microtips with filter barrier which may also be procured from Molbio REF 604072200 REF 604062010 respectively P owder free disposable gloves waste disposal container with lid SPECIMEN PREPARATION Truenat MTB requires purified nucleic acids from sputum specimen that are extracted using the Trueprep MAG Sample Prep Device and Trueprep MAG Sputum Sample Prep Kit Refer to the User Man
15. on the screen during the test run At the end of the test run a MTB DETECTED or NOT DETECTED result is displayed and in positive cases Ct values and colony forming units per milliliter cfu ml is also displayed on the screen Based on the Ctof the internal positive control IPC the validity of the test run is also displayed The IPC is a full process control that undergoes all the processes the specimen undergoes from extraction to amplification thereby validating the test run from sample to result Absence of or shift of PC Ct beyond a pre set range in case of negative samples invalidates the est run While IPC will co amplify in most positive cases also in some specimen having a high target oad the IPC may notamplify however the testrun is still considered valid The results can be printed via Bluetooth using the Truelab micro PCR printer or transferred to the lab computer or any remote computer via Wifi network or GPRS network Upto 5000 results can be stored on the analyzer for future recall and reference TARGET SELECTION The target sequence for this kitis part of the ribonucleoside diphosphate reductase gene the productof which provides the precursor for DNA synthesis The region selected is specific to the MTB complex CONTENTS OF THE Truenat MTB KIT A Individually sealed pouches each containing a 13 sear ity AND DECONTAMINATION 1 Truenat MTB chip based RealTi
16. s sputum samples containing a low load of MTB cells as characterized by smear microscopy and quantitative P CR were spiked with blood and pus upto 5 of the final sample concentration The DNA was extracted using Trueprep MAG Sputum Sample Prep Kit and Real Time PCR analysis was performed on the Truenat MTB The tests were carried outin duplicates Effectof interferences on detection of MTB using Truenat MTB Sample ID CtofS putum spiked CtofS putum spiked CtofControl sample with blood with pus withoutinterfering substances Average Ctvalues Average Ctvalues Sample 1 33 95 35 60 34 6 Sample 2 32 55 33 70 33 8 Sample 3 33 70 35 75 35 8 Sample 4 35 35 34 10 35 1 No specific inhibition was observed due to blood or pus in the Real Time PCR result using Truenat MTB 18 REFERENCES 1 WHO FactsheetMarch 2012 http www who int mediacentre factsheets fs104 en 2 Todar s Online Textbook of Bacteriology Kenneth Todar Ph D 3 WHO report2011 Global Tuberculosis Control 4 Karen R Steingart et al 2006 Sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis a systematic review The Lancet Infectious Diseases Volume 6 Issue 10 pp 664 674 5 P Famia et al 2002 Improving Sensitivity of Direct Microscopy for Detection of Acid FastBacilli in Sputum Use of Chitin in Mucus Digestion J Clin Microbiol 40 2 508 511 6 E O
17. ual of Trueprep MAG Sample Prep Device and the package insert of Trueprep MAG Sputum Sample P rep Kitfor details SAFETY PRECAUTIONS 1 Forinvitro diagnostic use only 2 Bring all reagents and specimen to room temperature 20 30 C before use 3 Donotuse kitbeyond expiry date 4 Carefully read the User Manuals and package inserts of all the components of the Truelab Real Time micro PCR System before use Allmaterials of human origin should be handled as though potentially infectious Do not pipette any material by mouth 7 Do noteat drink smoke apply cosmetics or handle contact lenses in the area where testing is oN done 8 Use protective clothing and wear disposable gloves when handling samples and while performing sample extraction PROCEDURAL PRECAUTIONS 1 Checkall packages before using the kit Damage to the packaging does not prevent the contents of the kitfrom being used However if the outer packaging is damaged the user must confirm that individualcomponents of the kit are intactbefore using them 2 Do notperform the testin the presence of reactive vapours e g from sodium hypochlorite acids alkalis or aldehydes ordust 3 While retrieving the Truenat MTB chip based RealTime PCR testand the DNase amp RNase free pipette tip from the pouch ensure that neither bare hands nor gloves that have been used for previous tests run are used PROCEDURAL LIMITATIONS 1 Optimal performance
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