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Reagent Pack for Processing Non-Sputum Samples for Extraction

with Sample Prep Kit for Sputum


Trueprep MAG Sputum - EPTB Sample Processing Pack (REF 60202SE50) is an add-on pack
containing 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 Time
Polymerase Chain Reaction (PCR) test for the quantitative detection and diagnosis of Mycobacterium
tuberculosis (MTB).
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). Over 80% of TB infections are pulmonary and the diagnosis is largely based on
analysis of sputum samples. However, when sputum production is not possible, Bronchoalveolar
Lavage (BAL) is used as a non-sputum sample. The remaining about 20% of TB infections is extra
pulmonary (EPTB) and occurs in various body fluids and tissues.
There is no good reference diagnostic standard for EPTB. Culture, Histopathology and Nucleic Acid
Amplification Tests (NAAT) such as PCR are the most commonly used direct diagnostic methods.
Culture and histopathology requires specialized and controlled laboratory facility and highly skilled
manpower and takes days to weeks to provide result. PCR, especially Real Time PCR is sensitive and
specific and provides results in a few hours. The Truelab Real Time micro PCR System and
Truenat 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 at
resource 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 Device together with
Trueprep MAG Sputum Sample 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 sputum
samples.
The EPTB pack allows for processing non-sputum specimen such as BAL and extra pulmonary
samples for further extraction by Trueprep Mag Sputum Sample Prep Kit.
Non-sputum samples are paucibacillary in nature characterized by very low loads of MTB bacilli and
samples such as tissue, pus, abscess etc. have non-homogeneous distribution of the bacteria. Also
these samples tend to have high levels of PCR inhibitors. Hence it is necessary to digest such complex
specimen to release the bacteria, concentrate to get better yields of the Bacilli and also discard
potentially inhibitory substances. The EPTB pack employs a combination of chemicals and an enzyme
reagent to achieve these objectives and to enable further extraction and purification of the bacterial
DNA using Trueprep Mag Sputum kit.
The material provided in the Trueprep MAG Sputum-EPTB Pack is sufficient for processing 50
samples.
60202SE50
50 T
Trueprep MAG Sputum - EPTB Pack contains the following reagents.
1. Liquefaction reagent: 7 ml (dropper bottle)
2. Reconstitution buffer: 10 ml
3. Enzyme reagent (ENZ) tube: 50 nos
Trueprep MAG Sputum - EPTB Pack is stable for one year from the date of manufacture if stored
between 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 C
5. Sterile 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. Truelab micro 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 may
also be procured from Molbio (REF 604072200 / REF 604062010 respectively), Powder free
disposable 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
1. INTENDED USE
2. INTRODUCTION
3. PRINCIPLE
4. PACK SIZE
5. MATERIAL PROVIDED
6. STORAGE AND STABILITY
7. MATERIALS REQUIRED BUT NOT PROVIDED WITH THE KIT
8. TYPE OF SPECIMENS, VOLUME NEEDED FOR TEST AND STORAGE
Fresh specimen must either be processed immediately as per sample procedure outlined in section on
Sample Processing Protocol, or stored frozen at -20 C. Frozen samples must be brought to room
temperature before starting sample processing.
Note: Frozen specimen should not be subjected to more than 3 freeze/thaw cycles as this can lead to
erroneous 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 Device and the package
insert of Trueprep MAG Sputum - EPTB Pack and Trueprep MAG Sputum Sample Prep Kit
before 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 performing
extraction.
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 that
components 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-contamination
between 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 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 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 to
local 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 after
decontamination either e.g.: 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).
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 CSF
1. 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 MAG
Sputum Kit.
B. Protocol For All Other Samples
1. 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 MAG
Sputum Kit.
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 Trueprep
Mag Sputum - EPTB pack, extracted with Trueprep Mag Sputum kit and Real Time PCR was run
on Truenat MTB chips and the results were compared to culture. There was an overall agreement of
82.17% between the two methods. 12 samples were culture positive and Truenat MTB negative
and 11 samples were Truenat MTB positive and culture negative. Three samples showed invalid
results on Truenat MTB and 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
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:
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In vitro Diagnostic Test.
Not for medicinal use.
IVD
Refer package insert
for instructions before use
Catalogue
Number
REF
Date of
Manufacture
Date of
Expiry
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