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Reagent Pack for Processing Non-Sputum Samples for Extractionwith Sample Prep Kit for Sputum

Trueprep MAG Sputum - EPTB Sample Processing Pack (REF 60202SE50) is an add-on packcontaining reagents for DNA extraction from non-sputum samples using Trueprep MAG Sputum,Sample Prep Kit for further Real Time PCR analysis on Truenat MTB, chip-based Real TimePolymerase Chain Reaction (PCR) test for the quantitative detection and diagnosis of Mycobacteriumtuberculosis(MTB).

Tuberculosis (TB) is an infectious disease caused predominantly by the bacillus Mycobacteriumtuberculosis. It typically affects the lungs (pulmonary TB) but can affect other sites as well (extrapulmonary TB). Over 80% of TB infections are pulmonary and the diagnosis is largely based onanalysis of sputum samples. However, when sputum production is not possible, BronchoalveolarLavage (BAL) is used as a non-sputum sample. The remaining about 20% of TB infections is extrapulmonary (EPTB) and occurs in various body fluids and tissues.There is no good reference diagnostic standard for EPTB. Culture, Histopathology and Nucleic AcidAmplification Tests (NAAT) such as PCR are the most commonly used di rect diagnostic methods.Culture and histopathology requires specialized and controlled laboratory facility and highly skilledmanpower and takes days to weeks to provide result. PCR, especially Real Time PCR is sensitive andspecific and provides results in a few hours. The Truelab Real Time micro PCR SystemandTruenat MTB, chip based Real Time PCR test for MTB makes real time PCR technology rapid,simple, robust and user friendly and offers sample to result capability in about one hour even atresource limited settings.The PCR process necessitates the extraction and purification of nucleic acids from clinical specimens,to free it from potential PCR inhibitors. The Trueprep MAG Sample Prep Devicetogether withTrueprep MAG SputumSample Prep Kit provides an easy method of nucleic acid extraction and

purification for further PCR on Truenat MTB. However, this kit is designed to handle only sputumsamples.The EPTB pack allows for processing non-sputum specimen such as BAL and extra pulmonarysamples for further extraction by Trueprep Mag SputumSample Prep Kit.

Non-sputum samples are paucibacillary in nature characterized by very low loads of MTB bacilli andsamples such as tissue, pus, abscess etc. have non-homogeneous distribution of the bacteria. Alsothese samples tend to have high levels of PCR inhibitors. Hence it is necessary to digest such complexspecimen to release the bacteria, concentrate to get better yields of the Bacilli and also discardpotentially inhibitory substances. The EPTB pack employs a combination of chemicals and an enzymereagent to achieve these objectives and to enable further extraction and purification of the bacterialDNA using Trueprep Mag Sputumkit.

The material provided in the Trueprep MAG Sputum-EPTB Pack is sufficient for processing 50samples.

60202SE50

50 T

Trueprep MAG Sputum- EPTB Pack contains the following reagents.1. Liquefaction reagent: 7 ml (dropper bottle)2. Reconstitution buffer: 10 ml3. Enzyme reagent (ENZ) tube: 50 nos

Trueprep MAG Sputum- EPTB Pack is stable for one year from the date of manufacture if storedbetween 2C to 30C. Avoid exposure to light. It is also stable for 4 weeks at temperatures up to 40 C.

1. Disposable micro pestle (for tissue homogenization)2. Microcentrifuge and tubes (2 ml)

3. Centrifuge and tubes (for upto10 ml volume)4. Dry bath at 60 C5. Steri le water 6. Truelab Real Time micro PCR Workstation(REF 603010001) consisting of

1. Trueprep MAG Sample Prep Device (REF 603040001).2. Truelab Uno Real Time micro PCR Analyzer (REF 603020001).3. Truelabmicro PCR Printer(REF 603050001).4. Truepet Precision Micropipettes- 6L, 50L, 100L, 500L, 1000L.

(REF 604010006 / 604020050 / 604030100 / 604040500 / 604051000).Also required additionally are:DNase and RNase-free pipette tips (2-200L / 200-1000L microtips) with filter barrier, which mayalso be procured from Molbio (REF 604072200 / REF 604062010 respectively), Powder freedisposable gloves, waste disposal container with Sodium Hypochlorite solution (0.5%).

Sl no Sample Type Sample volume

1 Bronchoalveolar Lavage (BAL) 10 ml

2 Pleural fluid, Peritonial fluid 10 ml

3 Cerebrospinal Fluid (CSF) 3 5 ml

4 Pus, Abscess 0.5 ml

5 Lymphnode aspirate 5 ml

6 Tissue 10 - 20 mg

INTENDED USE

INTRODUCTION

PRINCIPLE

PACK SIZE

MATERIAL PROVIDED

STORAGE AND STABILITY

MATERIALS REQUIRED BUT NOT PROVIDED WITH THE KIT

TYPE OF SPECIMENS, VOLUME NEEDED FOR TEST AND STORAGE

Fresh specimen must either be processed immediately as per sample procedure outlined in section onSample Processing Protocol, or stored frozen at -20 C. Frozen samples must be brought to roomtemperature before starting sample processing.Note: Frozen specimen should not be subjected to more than 3 freeze/thaw cycles as this can lead toerroneous results.

1. For in vitro diagnostic use only.0 0

2. Bring all reagents and specimen to room temperature (15 C - 30 C) before use.3. Do not use kit beyond expiry date.4. Read carefully the User Manual of Trueprep MAG Sample Prep Deviceand the package

insert of Trueprep MAG Sputum- EPTB Pack and Trueprep MAG SputumSample Prep Kitbefore use.

5. All materials of human origin should be handled as though potentially infectious.6. Do not pipette any material by mouth.

7. Do not eat, drink, smoke, apply cosmetics or handle contact lens in the area where testing is done.8. Use protective clothing and wear disposable gloves when handling samples and while performingextraction.

1. Do not exchange kit components from different lots.2. Check all packaging before using the kit. Damage to the packaging does not prevent the contents

of the kit from being used. However, if the outer packaging is damaged the user must check thatcomponents of the kit are intact before using them.

3. Do not perform the assay in the presence of reactive vapours (e.g., from sodium hypochlorite,acids, alkalis or aldehydes) or dust.

4. All pipetting steps should be performed with utmost care and accuracy. Cross-contaminationbetween reagents and samples may invalidate results.

5. Separate filter barrier tips should be used to pipette separate reagents.

1. Spills of potentially infectious material should be cleaned up immediately with absorbent papertissue and the contaminated area should be decontaminated with disinfectants such as 0.5%

freshly prepared sodium hypochlorite (10 times dilution of 5% sodium hypochlorite (householdbleach) before continuing work.

2. Sodium hypochlorite should not be used on an acid-containing spill unless the spill-area is wipeddry first. Materials used to clean spills, including gloves, should be disposed off as potentially bio-hazardous waste e.g. in a biohazard waste container.

1. Disinfect the solutions or waste containing biological samples before discarding them according tolocal instructions.

2. Samples and reagents of human and animal origin, as well as contaminated materials,disposables, neutralized acids and other waste materials must be discarded afterdecontamination either e.g.: by immersion in a freshly prepared 0.5 % of sodium hypochlorite for30 minutes (1 volume of 5% sodium hypochlorite for 10 volumes of contaminated fluid or water).

3. Do not autoclave materials or solutions containing sodium hypochlorite.4. Chemicals should be handled in accordance with Good Laboratory Practice and disposed off

according to local regulations.

A. Protocol For BAL, Pleural fluid, Peritoneal Fluid and CSF1. Take appropriate volume (Refer section 8) of the sample in a tube.2. Add 3 drops of Liquefaction buffer to the sample, Mix for 5 minutes and centrifuge at 4000 g for 5

mins.3. Aspirate 500 l supernatant using a micropipette. Discard rest of the supernatant and retain the

pellet in the tube.4. Dispense the 500 l supernatant back into the tube and re-suspend the pellet.5. Transfer contents to ENZ tube, mix well and incubate at 60 C for 20 minutes.6. Transfer all the contents to Sample Pretreatment Tube (SPT) of Trueprep MAG Sputum

Sample Prep Kit and proceed with Extraction Procedure (Section 14) of Trueprep MAGSputumKit.

B. Protocol For All Other Samples1. Take appropriate volume (Refer section 8) of the sample in a tube. Tissue samples must be

homogenized in 100 l of reconstitution buffer + 400 l sterile water.2. Add 3 drops of Liquefaction buffer to the sample, Mix for 5 minutes and centrifuge at 4000 g for 5

mins.3. Discard the supernatant and retain the pellet.4. Add 100 l of reconstitution buffer and re-suspend the pellet.5. Transfer contents to ENZ tube and make up volume to 0.5 ml mark on the tube with sterile water.

Mix well and incubate at 60 C for 20 minutes.6. Transfer all the contents to Sample Pretreatment Tube (SPT) of Trueprep MAG Sputum

Sample Prep Kit and proceed with Extraction Procedure (Section 14) of Trueprep MAGSputumKit.

Note- If un-dissolved tissue remains, transfer only the clear fluid to SPT tube

A total of 132 samples comprising BAL and EPTB specimen were processed using the TrueprepMag Sputum- EPTB pack, extracted with Trueprep Mag Sputumkit and Real Time PCR was runon Truenat MTBchips and the results were compared to culture. There was an overall agreement of82.17% between the two methods. 12 samples were culture positive and Truenat MTBnegativeand 11 samples were Truenat MTBpositive and culture negative. Three samples showed invalidresults on Truenat MTBand were excluded from the analysis.

1. WHO Fact sheet March 2012. http://www.who.int/mediacentre/factsheets/fs104/en/.2. Todar's Online Textbook of Bacteriology- Kenneth Todar, Ph.D.3. WHO report 2011 Global Tuberculosis Control.4. N.Kennedy et.al (1994) Polymerase Chain Reaction for Assessing Treatment Response in Patients

with Pulmonary Tuberculosis. The Journal of Infectious Diseases Vol. 170, No. 3, pp. 713-716.5. Data on file: Molbio Diagnostics Pvt. Ltd.

9. SAFETY PRECAUTIONS

10. PROCEDURAL PRECAUTIONS

11. CLEANING AND DECONTAMINATION

12. DISPOSAL AND DESTRUCTION

13. SAMPLE PROCESSING PROTOCOL

14. PERFORMANCE

15. REFERENCES

REF

Sputum

EPTB pack

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M 46-47, Phase III B, Verna Industrial Estate,Verna, Goa - 403 722, India. E-mail: sales@tulipgroup.com

Molbio Diagnostics Pvt. Ltd.

Manufactured by:

TPS-EPTB/1113/VER-01

LOTBatch Number/ Lot Number

Contains sufficientfor tests

Manufacturer

02 C

030 C Store between

02-30 C

In vitroDiagnostic Test.Not for medicinal use.IVD

Refer package insertfor instructions before use

CatalogueNumberREF

Date ofManufacture

Date ofExpiry

SYMBOL KEYS: