3. DNase & RNase free pipette tip.

4. Desiccant pouch. REF 601090005 601090020

B. Package Insert. 5T 20T

6. CONTENTS OF THE TrueprepTM AUTO Universal Sample Pre-treatment Pack (only for
HBV TrueprepTM AUTO users)
A. Lysis buffer.
Chip-based Real Time PCR Test for Hepatitis B Virus REF 60205AB05 60205AB20
B. Disposable transfer pipette (graduated).
5T 20T
1. INTENDED USE
Truenat™ HBV (REF 601090005/601090020) is a chip-based Real Time Polymerase Chain Reaction 7. STORAGE AND STABILITY
(PCR) test for the quantitative estimation of the Hepatitis B Virus (HBV) in human blood /serum/ Truenat™ HBV chip is stable for two years from the date of manufacture if stored between 2-30oC. It is
plasma specimen and aids in the diagnosis of infection with Hepatitis B Virus and in the estimation of also stable for upto six (6) months at temperatures up to 40o C and one (1) month at temperatures up to
viral load. Truenat™ HBV runs on the Truelab™ Real Time Quantitative micro PCR Analyzers. 45oC. Avoid exposure to light or elevated temperatures (above recommended levels). Do not freeze.
TrueprepTM AUTO Universal Sample Pre-Treatment Pack is stable for two years from the date of
2. INTRODUCTION manufacture if stored between 2-40°C. It is also stable for one (1) month at temperatures upto 45°C.
Hepatitis B infection is caused by the Hepatitis B Virus (HBV) that affects around two billion people
globally, and causes around 6,00,000 deaths each year. The infection, which can be acute or chronic, 8. MATERIALS REQUIRED BUT NOT PROVIDED WITH THE KIT
occurs in the liver. Acute infections are mostly asymptomatic but sometimes may cause symptoms Truelab™ Real Time micro PCR Workstation (REF 603010001/62301001/ 633010001/64301001)
such as jaundice, extreme fatigue, vomiting and abdominal pain. A chronic infection can develop into consisting of
cirrhosis of the liver and liver cancer. Transmission occurs from contact with infected blood or other 1. Trueprep™ MAG / AUTO Sample Prep Device (REF 603040001/603041001).
body fluids through perinatal, transfusion, injection and sexual routes and through close contact with 2. Truelab™ Uno / Truelab™ Uno Dx/Truelab™ Duo/Truelab™ Quattro Real Time micro
infected family members, especially in early childhood. Despite the availability of effective vaccination PCR Analyzer (REF 603020001/603021001/603022001/603023001).
and antiviral drugs, Hepatitis B remains a major global health problem. The most common methods of 3. Truelab™ micro PCR Printer (REF 603050001).
diagnosing Hepatitis B are serology based assays and molecular tests. The HBV DNA is detectable 4. Truepet™ SP fixed volume precision micropipette - 6 µl (REF 604060006).
about three weeks before the appearance of serological markers. HBV viral load quantitation by PCR 5. Truelab™ Microtube Stand (REF 603070001).
is very useful for treatment initiation decisions and treatment monitoring. However PCR or Real Time Also required additionally are: TrueprepTM MAG Blood Sample Prep Kit (REF 602010005/REF
PCR tests have so far been restricted to centralized reference laboratories as they require skilled 602010050) / TrueprepTM AUTO Universal Cartridge Based Sample Prep Kit (REF 60203AR05/REF
manpower and elaborate infrastructure. Also the turnaround time for results could take a few days. 60203AR25), Truenat™ Universal Control Kit (REF 601100008), DNase and RNase-free pipette tips
The Truelab™ Real Time micro PCR System enables decentralization and near patient diagnosis and with filter barrier which may also be procured from Molbio, Powder free disposable gloves, Nylon
treatment monitoring of Hepatitis B infection by making real time PCR technology rapid, simple, robust flocked swabs, urine collection cup, waste disposal container with lid.
and user friendly and offering “sample to result” capability even at resource limited settings. This is
TM
achieved through a combination of lightweight, portable, mains / battery operated Truelab™ Real 9. SPECIMEN PREPARATION FOR EXTRACTION WITH TRUEPREP MAG
Time micro PCR Analyzer and TrueprepTM MAG / AUTO Sample Prep Device and room Truenat™ HBV requires purified nucleic acids from whole blood/plasma collected in EDTA anticoagulant
TM TM
temperature stable Truenat™ micro PCR chips and TrueprepTM MAG /AUTO Sample Prep kits so or serum specimen that are extracted using the Trueprep MAG Sample Prep Device and Trueprep
that even the peripheral laboratories with minimal infrastructure and minimally trained technician can MAG Blood Sample Prep kit. (Refer to the User Manual of TrueprepTM MAG Sample Prep Device and
easily perform these tests routinely in their facilities and report PCR results in less than an hour. the package insert of TrueprepTM MAG Blood Sample Prep kit for details).
Moreover, with these devices PCR testing can also be initiated in the field level, on site.
Truenat™ HBV is a disposable, room temperature stable, chip-based Real Time PCR test with dried 10. SPECIMEN PREPARATION FOR EXTRACTION WITH TRUEPREPTM AUTO
MgCl2 in reaction well and freeze dried PCR reagents in microtube for Truenat™ HBV requires purified nucleic acids from whole blood/plasma collected in EDTA anticoagulant
TM
performing Real Time PCR test for quantitative estimation of the Hepatitis B or serum specimen that are extracted using the Trueprep AUTO Universal Cartridge based Sample
Virus and runs on the Truelab™ Real Time micro PCR Analyzer. It requires only Prep Device and TrueprepTM AUTO Universal Cartridge based Sample Prep kit. Sample must be pre-
six (6) μL of purified DNA to be added to the reaction well for the analysis. The treated using TrueprepTM AUTO Universal Sample Pre-treatment pack. Transfer 250µl of whole blood
intelligent chip also carries test and batch related information including standard or 500µl of plasma/serum specimen using the transfer pipette provided into the Lysis buffer tube
values for quantitation. The TruenatTM HBV chip also stores information of used provided and mix well.
test to prevent any accidental re-use of the test. Sample Storage and Transportation:
NOTE : Truelab™ Uno / Truelab™ Uno Dx / Truelab™ Duo / Truelab™ Sample Pre-treatment decontaminates the specimen and makes it ready for storage/ transportation/
Quattro / Trueprep™ MAG / Trueprep™ AUTO / Truepet™ / Truenat™ are extraction. The specimen in this form is stable for up to 3 days at 40ºC and 1 week at 30ºC.
all registered trademarks of Molbio Diagnostics (P) Limited. Nucleic acid extraction: Use entire content from the Lysis Buffer tube containing specimen for
The Truelab™ Real Time micro PCR Analyzer is protected by the following patents and patents further procedure with the Trueprep™ AUTO Universal Cartridge Based Sample Prep Device and
pending: IN 2313/CHE/2007, WO 2009/047804 and corresponding claims of any foreign Trueprep™ AUTO Universal Cartridge Based Sample Prep Kit. (Refer to the User Manual of
counterpart(s) thereof. TrueprepTM AUTO Universal Cartridge Based Sample Prep device and the package insert of
The Truenat™ micro PCR chip is protected by the following patents and patents pending: IN TrueprepTM AUTO Universal Cartridge Based Sample Prep kit for details). Dispose off lysis buffer
2312/CHE/2007, WO 2009/047805 and corresponding claims of any foreign counterpart(s) tube and transfer pipette after use, as per the section on “Disposal and Destruction” (Section 18).
thereof.
11. SAFETY PRECAUTIONS
3. PRINCIPLE OF THE TEST 1. For in vitro diagnostic use only.
Truenat™ HBV works on the principle of quantitative Real Time Polymerase Chain Reaction based on 2. Bring all reagents and specimen to room temperature (20°C - 30°C) before use.
Taqman chemistry. The DNA from the patient sample is first extracted using Trueprep™ MAG Sample 3. Do not use kit beyond expiry date.
Prep Device and Trueprep™ MAG Blood Sample Prep Kit or using Trueprep™ AUTO Universal 4. Carefully read the user manuals and package inserts of all the components of the Truelab™
Cartridge based Sample Prep Device and Trueprep™ AUTO Universal Cartridge based Sample Prep Real Time micro PCR System before use.
Kit. The Truenat™ HBV chip is placed on the chip tray of the Truelab™ Real Time micro PCR 5. All materials of human origin should be handled as though potentially infectious.
Analyzer. Six (6) µL of the purified DNA is then dispensed using the provided micropipette and tip into 6. Do not pipette any material by mouth.
the microtube containing freeze dried PCR reagents and allowed to stand for 30-60 seconds to get a 7. Do not eat, drink, smoke, apply cosmetics or handle contact lenses in the area where testing is
clear solution. No mixing by tapping, shaking or by reverse pipetting should be done. Six (6) µL of done.
this clear solution is then pipetted out using the same pipette and tip and dispensed into the reaction 8. Use protective clothing and wear disposable gloves when handling samples and while
well of the Truenat™ HBV chip and the test is started. A positive amplification causes the dual labeled performing sample extraction.
fluorescent probe in the Truenat™ HBV chip to release the fluorophores in an exponential manner
and the emitted light is then captured by the built-in opto-electronic sensor and displayed as 12. PROCEDURAL PRECAUTIONS
amplification curve on the analyzer screen, on a real time basis during the test run. The Cycle threshold 1. Check all packages before using the kit. Damage to the packaging does not prevent the contents
(Ct) is defined as the number of amplification cycles required for the fluorescent signal to cross the of the kit from being used. However, if the outer packaging is damaged the user must confirm that
threshold (i.e. exceed the background signal). Ct levels are inversely proportional to the amount of individual components of the kit are intact before using them.
target nucleic acid in the sample. (i.e. the lower the Ct level the greater is the amount of target nucleic 2. Do not perform the test in the presence of reactive vapours (e.g., from sodium hypochlorite,
acid in the sample). In the case of negative samples, amplification does not occur and a horizontal acids, alkalis or aldehydes) or dust.
amplification curve is displayed on the screen during the test run. At the end of the test run, a HBV 3. While retrieving the Truenat™ HBV micro PCR chip, microtube and the DNase & RNase free
“DETECTED” or “NOT DETECTED” result is displayed and in positive cases, Ct values and pipette tip from the pouch, ensure that neither bare hands nor gloves that have been used for
International Units (IU) per milliliter (IU/ml) is also displayed on the screen. Based on the Ct of the previous tests run are used.
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 13. PROCEDURAL LIMITATIONS
thereby validating the test run from sample to result. Absence of or shift of IPC Ct beyond a pre-set 1. Optimal performance of this test requires appropriate specimen collection, handling, storage and
range in case of negative samples invalidates the test run. While IPC will co-amplify in most positive transport to the test site.
cases also, in some specimen having a high target load, the IPC may not amplify, however the test run 2. Though very rare, mutations within the highly conserved regions of the target genome where the
is still considered valid. The results can be printed via Bluetooth using the Truelab™ micro PCR printer Truenat™ assay primers and/or probe bind may result in the under-quantitation of or a failure to
or transferred to the lab computer/or any remote computer via Wifi network or 3G/GPRS network. Upto detect the presence of the concerned pathogen.
5000 results in TruelabTM Uno to 20000 results in TruelabTM Uno Dx/Duo/Quattro can be stored on 3. The instruments and assay procedures are designed to minimize the risk of contamination by
the analyzer for future recall and reference. PCR amplification products. However, it is essential to follow good laboratory practices and
ensure careful adherence the procedures specified in this package insert for avoiding nucleic
4. TARGET SELECTION acid contamination from previous amplifications, positive controls, or specimens.
The target sequence for this kit is part of the core/pre-core region of HBV genome. The region selected 4. A specimen for which the Truenat™ assay reports “Not Detected” cannot be concluded to be
is specific to HBV and conserved across the HBV Genotypes. 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.
5. CONTENTS OF THE Truenat™ HBV KIT
A. Individually sealed pouches, each containing 14. CLEANING AND DECONTAMINATION
1. Truenat™ HBV micro PCR chip. 1. Spills of potentially infectious material should be cleaned up immediately with absorbent paper
2. Microtube with freeze dried PCR reagents. 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 cross-reactivity in the Truenat™ HBV assay. No interference in the performance of the Truenat™
bleach) before continuing work]. HBV assay was observed with the listed group of organisms.
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.
Escherichia coli
15. TEST PROCEDURE Chlamydia trachmatis Streptococcus mutans Herpes Simplex virus
(Please also refer the TruelabTM Real Time micro PCR Analyzer user manual) Gardnerella vaginalis
1. Switch on the Truelab™ Analyzer. Virus
Adenovirus
2. If using the TruelabTM Uno device, also switch on the touch screen. If using the TruelabTM Uno Epstein-Barr virus
Dx/Duo/Quattro proceed to step 3.
3. Select User and enter password. Linearity & Assay range:
The linearity assay was performed according to CLSI HBV Linearity Curve y=-2.647x + 36.96
4. For TruelabTM Uno/Uno Dx, select the test profile for “HBV” to be run from the Profiles Screen on the Guidelines. Serial dilutions of the HBV DNA cloned in 40
R2 = 0.998

Analyzer screen. For TruelabTM Duo/Quattro, select the Bay (Idle1/2) for Duo and (Idle1/2/3/4) for a plasmid was made from 5.09E+09 copies/ml to 35
Quattro from the Status Screen to view the Profiles Screen. Select the test profile for “HBV” to be run 5.09E+02 copies/ml were made and nucleic acids
30
25
from the Profiles Screen on the Analyzer screen. were extracted on TrueprepTM AUTO sample prep

Ct
20
5. Enter the patient details as prompted in the Truelab™ Analyzer screen. device in triplicates for each dilution followed by PCR 15

6. Press Start Reaction. on TruelabTM Uno Dx PCR device. The assay is found 10
5
7. For TruelabTM Uno/Uno Dx, Press the eject button to open the chip tray. For TruelabTM Duo/Quattro, to be linear over 8 orders of magnitude (from 5.09E+09 0

the chip tray opens automatically on tapping the “Start Reaction” button. copies/ml to 5.09E+02 copies/ml ) for HBV DNA 0 2 4 6
Log copies/mL
8 10

8. Open a pouch of Truenat™ HBV and retrieve the micro PCR chip, microtube and DNase & RNase
free pipette tip. Limit of detection (Analytical Sensitivity):
The LOD was determined by testing dilutions of HBV
9. Label the chip with the patient ID using a marker pen at the space provided on the back side of the 4th International Standard from NIBSC. Probit analysis
chip. of the data was used to determine the concentration of
10. Place the Truenat™ HBV chip on the chip tray without touching the white reaction well. The reaction the respective DNA with 95% probability. LOD was
well should be facing up and away from the Analyzer. Gently press the chip to ensure that it has determined to be 55.92 IU/ml for HBV.
seated in the chip tray properly.
11. Place the microtube containing freeze dried PCR reagents in the microtube stand provided along with
the TruelabTM Real Time micro PCR workstation after ensuring that white pellet of dried PCR
reagents remains at the bottom of the microtube. Remove the microtube cap and dispose it off as
per the section on “Disposal and Destruction” (Section 18). Using the filter barrier tip provided in the Robustness:
pouch, pipette out six (6) µL of the purified DNA from the Elute Collected Tube into the microtube. To determine whether the Truenat™ HBV chip-based Real Time PCR test showed any signs of carryover
Allow it to stand for 30-60 seconds to get a clear solution. Do not mix it by tapping, shaking or by of PCR products between runs, alternating runs of positive samples and negatives samples were
reverse pipetting. Using the same filter barrier tip, pipette out six (6) µL of this clear solution and performed. 10 positive samples and 10 negative samples were used for the study. The Truenat™ HBV
dispense into the centre of the white reaction well of the Truenat™ HBV chip. Take care not to scratch test did not exhibit detectable carryover contamination between positive to negative sample runs.
the internal well surface and not to spill elute on the outside of the well. Dispose off the microtip as per
the section on “Disposal and Destruction” (Section 18). Reproducbility:
12. For TruelabTM Uno/Uno Dx, slide the chip tray containing the Truenat™ HBV chip-based Real Time The reproducibility of TruenatTM HBV assay was determined between three different users and between
PCR test loaded with the sample into the TruelabTM Analyzer. Press Done on the “Please Load three different devices. Three different titres of samples (High, Medium and Low) were used for this
Sample” Alert message. For TruelabTM Duo/Quattro, select “YES” at the Please load Sample study. The variation in the standard deviation between the users and devices were calculated. The
prompt. Chip tray will close automatically and the reaction will start. standard deviation values obtained for both three user study and three device variation study was within
13. Read the result from the screen. the accepted range of =<1.5 Ct.
14. After the reaction is completed, for TruelabTM Uno/Uno Dx, push the Eject button to eject the chip tray.
For TruelabTM Duo/Quattro, tap the “Open/Close Tray” button to eject the chip tray. Interference:
The purpose of this study is to determine the effect of potentially interfering substances on the Truenat™
15. Take out the Truenat™ HBV micro PCR chip at end of the test and dispose it off as per the section on HBV assay. The experiments were performed with HBV positive samples spiked in negative human
“Disposal and Destruction” (Section 18). plasma. Interfering substances used in this study are: Albumin- 9g/dL, Triglycerides-3.0 mg/dL, Human
16. Turn on Truelab™ micro PCR printer and select print on the screen for printing out hard copy of the DNA-0.4 mg/dL and Hemoglobin-500 mg/dL. The presence of these substances did not interfere with the
results. Test results are automatically stored and can be retrieved any time later. (Refer to TruelabTM performance of TruenatTM HBV assay. The standard deviation values obtained for both three user study
Analyzer manual). and three device variation study was within the accepted range of =<1.5 Ct.
17. Switch off the TruelabTMAnalyzer.
Accuracy of TruenatTM HBV assay:
16. RESULTS & INTERPRETATIONS Accuracy was determined by performing DNA extractions and Truenat™ HBV PCR for varying titres of
Two amplification curves are displayed on the Truelab™ Real Time micro PCR Analyzer screen when samples over 5 consecutive days. The standard deviation values obtained were within the accepted
optical plot is selected to indicate the progress of the test. Both the target and the internal positive range of =<1.5 Ct for HBV.
control (IPC) curves will take a steep, exponential path when the fluorescence crosses the threshold
value in case of positive samples. The Ct will depend on the number of viral genomes in the sample. Precision of TruenatTM HBV assay:
The target curve will remain horizontal throughout the test duration and the IPC curve will take an Precision was tested by performing Truenat™ HBV assay of High, Medium and Low titre HBV DNA for
exponential path in case of negative samples. In case the IPC curve remains horizontal in a negative five consecutive days. Every day PCR for each titre DNA was run in duplicates. The standard deviation
sample, the test is considered as Invalid. At the end of the test run, the results screen will display values obtained were within the accepted range of =<1.5 Ct for TruenatTM HBV assay.
“DETECTED” for Positive result or “NOT DETECTED” for Negative result. The result screen would
also display the Ct value and the IU/ml for positive specimen. The result screen also displays the Analytical reactivity or Inclusivity:
validity of the test run as “VALID” or “INVALID”. Invalid samples have to be repeated with fresh Analytical reactivity or Inclusivity of TruenatTM HBV assay was performed on a clinical; genotype panel
specimen from the sample preparation stage. While IPC will co-amplify in most positive cases also, in consisting of 6 HBV Genotypes. The genotype panel was procured from Discovery life Sciences (DLS).
some specimen having a high target load, the IPC may not amplify, however the test run is still The respective genotype DNA was extracted using the TrueprepTM AUTO sample prep device in
considered valid. duplicates followed by PCR on TruelabTM Uno Dx PCR analyzer.

17. QUALITY CONTROL PROCEDURES 20. REFERENCES

To ensure that the Truelab™ Real Time micro PCR Analyzer is working accurately, run positive and (1).http://www.who.int/mediacentre/factsheets/fs204/en/.(2).http://www .cdc.gov/hepatitis/hbv/pdfs/hepbg
negative controls from time to time. The Universal Control Kit containing Positive Control and Negative eneralfactsheet.pdf. (3). Fryer JF, Heath AB, Wilkinson DE, Minor PD and the collaborative study group.
Control must be ordered separately. It is advisable to run controls under the following circumstances: Collaborative study to evaluate the proposed 3rd WHO International Standard for hepatitis B virus (HBV) for
● Whenever a new shipment of test kits is received. ● When opening a new test kit lot. ● If the nucleic acid amplification technology (NAT)-based assays. WHO ECBS Report 2011;.2170 .(4).Abe, Aki, et
al. Quantitation of hepatitis B virus genomic DNA by real-time detection PCR. Journal of Clinical
temperature of the storage area falls outside of 2-30o C. ● By each new user prior to performing testing
Microbiology 37.9 (1999): 2899-2903. (5).Chen, Ren Wei, et al. Realtime PCR for detection and quantitation
on clinical specimen.
of hepatitis B virus DNA. Journal of medical virology 65.2 (2001): 250-256. (6). Brechtbuehl, K., et al. A rapid
real-time quantitative polymerase chain reaction for Hepatitis B virus. Journal of virological methods 93.1
18. DISPOSAL AND DESTRUCTION
(2001): 105-113.
1. Submerge the used Truenat™ HBV chip, microtube, microtube cap, transfer pipette, pipette tips,
lysis buffer tube etc. in freshly prepared 0.5% sodium hypochlorite solution for 30 minutes before SYMBOL KEYS
Consult In vitro Diagnostic Medical Temperature
For
disposal as per the standard medical waste disposal guidelines. instructions IVD Device. Not for medicinal
Limitation REF Catalogue
Number 2 single This Side
Up
Manufacturer
for use use. use only
2. Disinfect the solutions and/or solid waste containing biological samples before discarding them
according to local regulations. Date of Date of
LOT
Batch Number
Caution Contains sufcient EC REP
Authorised Representative in
the European Community
Manufacture Expiry / Lot Number for <n> tests
3. 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).
4. Do not autoclave materials or solutions containing Sodium hypochlorite. Molbio Diagnostics Pvt. Ltd.
5. Chemicals should be handled in accordance with Good Laboratory Practice and disposed off
according to the local regulations. H. No. 13, Sagar Society, Dona Paula, Plot No. L-46, Phase II D,
TNHBV/0818/V-01

Panaji, North Goa, Goa - 403004, INDIA Verna Industrial Estate, Verna,
19. SPECIFIC PERFORMANCE CHARACTERISTICS www.molbiodiagnostics.com Goa - 403 722, INDIA
Analytical Exclusitivity (Primer specificity): The following microorganisms were evaluated in silico
from the NCBI database using the NCBI nucleotide blast and primer blast tools to determine potential EC REP Qarad b.v.b.a. Cipalstraat 3, B-2440 Geel, Belgium