For professional use only

HBV PCR detection Kit

USER MANUAL

"DNA-Technology, Research & Production" LLC

Russia, 142281, Moscow Region,

Protvino, 20 Zheleznodorozhnaya Street,

Phone/fax: +7(495)640.17.71

+7(4967)31.06.70,

E-mail: [email protected], [email protected]

Customer service department: +7(495)640.16.93

E-mail: [email protected],

www.dna-technology.ru

R1-P602-23/9EU F1-P602-21/1EU
R1-P602-S3/9EU E1-P602-50/1EU 184-8.2024.04.22
R1-P602-24/9EU E1-P602-20/1EU
F1-P602-51/1EU
 TABLE OF CONTENTS

1. INTENDED USE 4

2. METHOD 4

3. CONTENT 5

4. REAGENTS AND EQUIPMENT REQUIRED BUT NOT PROVIDED 6

5. WARNINGS AND PRECAUTIONS 7

6. DNA EXTRACTION PROTOCOL 7

7. PCR PROTOCOL 9

8. CONTROLS 11

9. DATA ANALYSIS 12

10. TROUBLESHOOTING 13

11. STORAGE AND HANDLING REQUIREMENTS 13

12. SPECIFICATIONS 14

13. QUALITY CONTROL 14

14. KEY TO SYMBOLS 14

Annex А 15

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 1. INTENDED USE

The HBV PCR detection Kit is intended for research and diagnostic applications. The HBV PCR detection
Kit is an in vitro Nucleic Acid Test (NAT) based pathogen detection product. The HBV PCR detection Kit is
designed to detect Hepatitis B Virus (HBV) nucleic acids in human blood plasma.

The HBV PCR detection Kit can be used in clinical practice and for research purposes for HBV diagnostics.

2. METHOD

The implemented PCR method is based on amplification of a target DNA sequence.

The detection can be performed in each of three variants: real-time (HBV Real-Time PCR Detection Kit),
endpoint (HBV FLASH PCR Detection Kit) and HBV Сonventional PCR Detection Kit.

The HBV Real-Time PCR Detection Kit is based on fluorescent modification of the PCR method. The PCR-
mix contains target-specific probes bearing reporter and quencher molecules. Once hybridized to a target
sequence, the probes become activated. As a result of activation fluorescence increases proportionally to
target sequence amplification. The intensity of fluorescence is measured at every cycle of reaction with a
Real-time PCR thermal cycler data collection unit and analyzed with the software provided. The HBV
FLASH PCR Detection Kit is based on the same principle but the fluorescence is measured only once after
reaction. HBV Сonventional PCR Detection Kit is developed for PCR result detection by electrophoresis in
the agarose gel.

The automatic analysis available on “DNA-Technology” made instruments: DTlite or DTprime REAL-TIME
Thermal Cyclers for HBV Real-Time PCR Detection Kit (see the catalogue at www.dna-technology.ru/en to

see available supply options) and Gene or Gene-4 Fluorescence Readers О-GENE-EU, О-GENE4-EU
for HBV FLASH PCR Detection Kit.

The HBV Real-Time PCR Detection Kit is also approved for use with iQ (Bio-Rad Laboratories) and
Rotor-Gene Q (Qiagen) real-time thermal cyclers. The HBV FLASH PCR Detection Kit is also approved for
use with Ala1/4 fluorescence reader (BioSan).

DNA extraction. On this step the internal control sample (IC) is added to the samples. It is needed for test
quality assurance.

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 3. CONTENT

Table 1. PREP-NA DNA/RNA Extraction Kit1

Reagent Description Total volume Amount

Lysis buffer Colorless, soapy liquid 30 mL 1 vial

Precipitation buffer Colorless liquid 40 mL 1 vial

Washout solution 1 Colorless liquid 50 mL 1 vial

Washout solution 2 Colorless liquid 30 mL 1 vial
5.0 mL
Dilution buffer Colorless liquid 4 tubes
(1.25 mL in each tube)
3.0 mL
Negative control (C-) Colorless liquid 2 tubes
(1.5 mL in each tube)
Internal control (DNA-IC) Colorless liquid 1.0 mL 1 tube
Internal control (RNA-IC) Colorless liquid 1.0 mL 1 tube

Table 2. HBV PCR detection Kit

Reagent Description Total volume Amount

96 or 100 separate
Colorless liquid and 1.92 mL or 2.0 mL
Paraffin sealed PCR-mix or stripped tubes
white waxy fractions (0.02 mL per tube)
of 0.2 or 0.5 mL

TECHNO Taq-polymerase Colorless viscous liquid 50 µL 1 tube

1 mL
PCR-buffer Colorless liquid 2 tubes
(0.5 mL in each tube)

Positive control Colorless liquid 150 µL 1 tube

Mineral oil (not supplied in 2.0 mL

Colorless viscous liquid 2 tubes
Kit for Rotor-Gene Q) (1.0 mL in each tube)

Accessories:
Caps for strips 12 pieces2.
The approximate total time needed to perform the assay from 4 hours (including sample preparation).

Upon customer’s request, optional supply of a reagent kit for DNA electrophoretic detection is possible,
including:
- Electrophoresis mix (9.55 g) and Agarose gel (5 plates)

The PREP-NA DNA/RNA Extraction Kit is sufficient for extraction of 100 samples.

The HBV PCR detection Kit sufficient to test 96/100 samples including negative and positive controls.

1
- can be included into the kit if requested.
2
- in case of using stripped tubes.

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 4. REAGENTS AND EQUIPMENT REQUIRED BUT NOT PROVIDED

4.1 Specimen collection

The whole blood samples shoud be collected in 2.0 or 4.0 mL Vacuette type tubes with EDTA in 2.0 mg/mL
final concentration. The sodium citrate anticoagulant is also applicable.

The use of heparin anticoagulant is not allowed.

4.2 DNA extraction and PCR

Biological (microbiological) safety cabinet class II;

UV PCR cabinet;
Vortex mixer;
0.2, 0.5 and 1.5 mL tubes;
PCR tube rack for 0.2, 0.5 and 1.5 mL tubes;
Vacuum blood collection tubes (Vacuette for example), containing ethylenediaminetetraacetic acid
disodium salt (EDTA) or sodium citrate anticoagulant;
Аspirator with trap flask to remove supernatants;
Single channel pipettes (volume range 0.5-10 µL, 5-40 µL, 40-200 µL, 100-1000 µL);
RNase and DNase free filtered pipette tips (volume range 20 µL, 50 µL, 200 µL, 1000 µL);
Powder-free surgical gloves;
Disinfectant solution;
Container for used pipette tips;
Household refrigerator with a freezer chamber;
High speed centrifuge (RCF 16 000 x g);
Thermostat (temperature range 40-95 °C);
Real-time PCR thermal cycler (for HBV Real-Time PCR Detection Kit);

Tercyc Conventional PCR Thermal Cycler ( О-TP4-EU) or equivalent (for HBV FLASH PCR
Detection Kit and HBV Conventional PCR Detection Kit );

Gene or Gene-4 Fluorescence Reader ( О-GENE-EU, О-GENE4-EU) or Ala1/4 fluorescence reader

or equivalent (for HBV FLASH PCR Detection Kit);

For electrophoretic detection (for HBV Conventional PCR Detection Kit):

• AC power supply;
• electrophoretic chamber;
• transilluminator;
• 1.0 L volumetric flask;
• distilled water;
• 1.0 mm diameter steel wire.

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 5. WARNINGS AND PRECAUTIONS

The laboratory makeup should comply the requirements regulating work with microorganisms of
I-IV classes of pathogenicity.
Handle and dispose all biological samples, reagents and materials used to carry out the assay as if they
were able to transmit infective agents. Avoid direct contact with the biological samples reagents and
materials used to carry out the assay. Any material coming in contact with the biological samples must be
treated for at least 30 minutes with disinfecting solution or autoclaved for 1 hour at 121 °С before disposal.
Molecular biology procedures, such as nucleic acids extraction, reverse transcription, amplification and
detection require qualified staff to avoid the risk of erroneous results, especially due to the degradation
of nucleic acids contained in the samples or sample contamination by amplification products.
All oligonucleotide components are produced by artificial synthesis technology according to internal
quality control protocol and do not contain blood or products of blood processing.
Positive control is produced by artificial DNA synthesis technology. Positive control does not include parts
of infectious agents.
All the liquid solutions are designed for single use and cannot be used more than once in amplification
reactions. Plastic tubes do not contain phthalates. Do not breathe gas/fumes/vapour/spray produced by
the components of the kit. Do not eat/drink components of the kit. Avoid contact with eyes. Do not use
the kit after the expiry date provided. Only use the reagents provided in the kit and those recommended
by manufacturer. Do not mix reagents from different batches. Do not use reagents from third party
manufacturers’ kits.
Significant health effects are NOT anticipated from routine use of this kit when adhering to the
instructions listed in the current manual.

6. DNA EXTRACTION PROTOCOL

The HBV PCR detection Kit is designed to detect DNA extracted from blood plasma. Shake the tube
containing blood sample thoroughly to mix the blood and anticoagulant.

Using of heparin as anticoagulant is not allowed.

The overall storage of the sample should not exceed 6 hours.

The transportation and storage temperature from collecting the sample till analysis should be in between
2 °С and 8 °С range.

Whole blood cannot be frozen.

6.1 To obtain the plasma spin the tubes with blood at 800-1600 х g (corresponds to 3000 rpm on
Eppendorf Centrifuge 5424) for 20 min at room temperature (between 18 °С and 25 °С).

Relative centrifugal force (RCF or g) depends on rotation frequency and centrifugation radius
(Annex А). To establish if your centrifuge meets the requirements apply to the exploitation manual for
centrifuge.
6.2 Take the upper fraction (plasma) with an automatic sampler and put it into the new 1.5 mL
tube. The blood plasma can be stored at temperature from minus 18 °С to minus 22 °С for
no longer than 3 months.

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 The lysis buffer can contain the precipitate. Dissolve it at 65 °С for 10 min. prior to use.

At this step of assay use only disposable pipette tips witch have filter and are RNase and DNase free.

6.3 Mark the required number of 1,5 mL tubes by the following scheme: for each test sample
and for negative control (C-).
For example: if you need to test 10 samples, mark 11 tubes (10 for the samples, 1 for C-).

6.4 Add 10 µL of the premixed internal control (DNA-IC) in each tube.

6.5 Add 300 µL of the lysis buffer avoiding contact of the pipette tip with an edge of the tube.
Close the tubes.

Open the tube, add sample, then close the tube before proceeding to the next DNA sample to
prevent contamination.

6.6 Add 100 µL of the premixed blood plasma sample into the marked tubes. Do not add
samples to the “C-“ tube.
6.7 Add 100 µL of the “C-“ into corresponding tube.
6.8 Close the tubes and mix them for 3–5 sec twice and spin down the drops for 3–5 sec
at room temperature.
6.9 Incubate the tubes for 15 min at 65 °С, spin down the drops at 16000 x g (corresponds to
13000 rpm on Eppendorf Centrifuge 5424) for 30 sec at room temperature.
6.10 Add 400 µL of the precipitation buffer into all tubes. Close the tubes and mix them for
3-5 sec twice.
6.11 Spin the tubes at 16000 x g (corresponds to 13000 rpm on Eppendorf Centrifuge 5424) for
15 min at room temperature.
6.12 Remove the supernatant avoiding contact of the pipette tip with the precipitate. Use new
tip for each sample.
6.13 Add 500 µL of the washout solution №1 to the precipitate and invert the tubes gently 3-5 times.
6.14 Spin the tubes at 16000 x g (corresponds to 13000 rpm on Eppendorf Centrifuge 5424) for
5 min at room temperature.
6.15 Remove the supernatant avoiding contact of the pipette tip with the precipitate. Use new
tip for each sample.
6.16 Add 300 µL of the washout solution №2 to the precipitate and invert the tubes gently 3-5 times.
6.17 Spin the tubes at 16000 x g (corresponds to 13000 rpm on Eppendorf Centrifuge 5424) for
5 min at room temperature.
6.18 Remove the supernatant avoiding contact of the pipette tip with the precipitate. Use new
tip for each sample.
6.19 Open the tubes and dry the precipitate at 65 °С for 5 min.
6.20 Add 25 µL of the dilution buffer to the precipitate. Spin down the drops for 3–5 sec.
6.21 Incubate the tubes for 10 min at 65 °С.
6.22 Spin down the drops at 16000 x g (corresponds to 13000 rpm on Eppendorf Centrifuge
5424) for 30 sec.
The DNA preparation is ready for PCR amplification.
The DNA preparation can be stored at temperature from minus 18 °С to minus 22 °С for no longer than
1 month or at temperature from minus 68 °С to minus 72 °С for no longer than 1 year.
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 7. PCR PROTOCOL

7.1 Mark tubes with PCR-mix for each test sample, negative control (C-), positive control (C+).
Mark additionally two tubes for background buffer (applicable to FLASH PCR kits).
For example if you need to test 10 samples, mark 12 tubes (10 for samples, 1 for C-,
1 for C+). For FLASH PCR kit mark 14 tubes (10 for samples, 1 for C-, 1 for C+ and
2 for background buffer).

Mark only the caps of the tubes when using Rotor-Gene Q Thermal Cycler.
7.2 Thaw PCR-buffer at the room temperature.
7.3 Mix the PCR-buffer and TECHNO Taq-polymerase thoroughly (3-5 sec), then spin briefly
(1-3 sec) at room temperature.

Hold TECHNO Taq-polymerase at room temperature as short time as possible. The overheating is
detrimental to its performance.
7.4 Prepare the mixture of PCR-buffer and TECHNO Taq-polymerase (TECHNO Taq-polymerase
solution). Add into the one tube:
• 10 х (N+1) µL of PCR-buffer;
• 0.5 × (N+1) µL of TECHNO Taq-polymerase;
N — number of the marked tubes including C-, C+, background tubes.
For example if you need to test 10 samples (12 marked tubes), prepare mixture of PCR-buffer
and TECHNO Taq-polymerase for 13 (12+1) tubes: 130 µL PCR-buffer + 6.5 µL TECHNO Taq-polymerase.
7.5 Vortex the tube with TECHNO Taq-polymerase solution for 3-5 sec and spin down the
drops for 1-3 sec at room temperature. The maximum storage time for prepared mixture
at the temperature between 2 °С and 8 °С for no longer than 1 hour.
7.6 Add 10 µL of TECHNO Taq-polymerase solution into each tube (except background tubes).
Add 10 µL of background buffer into corresponding tubes (applicable to FLASH PCR kits).
Avoid paraffin layer break.
7.7 Add one drop (~20 µL) of mineral oil into each tube (not applicable to kits approved for
use with Rotor-Gene Q thermal cycler). Close tubes tightly.
7.8 Vortex the tubes with samples for 3-5 sec and spin down the drops for 1-3 sec.
7.9 Vortex the tubes with DNA for 3-5 sec and spin down the drops for 1-3 sec at room
temperature.
7.10 Add 5.0 µL of DNA sample into corresponding tube. Avoid paraffin layer break. Do not add
DNA into the C-, C+ and background (applicable to FLASH PCR kits) tubes. Avoid paraffin
layer break.

Open the tube, add DNA sample, then close the tube before proceeding to the next DNA sample
to prevent contamination. Use filter tips.
7.11 Add 5.0 µL of C- which has passed DNA isolation stage into C- and background (applicable
to FLASH PCR kits) tubes. Add C+ into corresponding tube. Avoid paraffin layer break.
7.12 Spin tubes briefly (1-3 sec) at room temperature (not applicable to kits approved for use
with Rotor-Gene Q thermal cycler).
7.13 Set the tubes to the Thermal Cycler.
Launch the Thermal Cycler software and run PCR according to instructions supplied with
device, considering 35 µL reaction mix volume. See tables 3-7 to refer the cycling program and
table 8 to refer the detection channels (applicable to Real-Time PCR kits). Using Tercyc cycler
you need to choose «Fast active regulation» regulation algorithm.
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Table 3. The PCR program for Tercyc Conventional PCR Thermal Cycler (applicable to Conventional PCR
kits and FLASH PCR kits)
Step For thermal cyclers with active regulation Number of cycles
Temperature, °C Time
min sec
1 94 5 0 1

2 94 0 10 50
62 0 20

3 10 … … Storage
Note! When working with FLASH PCR detection Kits once prepared and amplified “BACKGROUND” tubes
may be used many times at each PCR results detection with reaction tubes from the same lot.
“BACKGROUND” tubes can be stored at temperatures between 2 °С and 8 °С and out of light for no longer
than 1 month. During the detection procedure “BACKGROUND” tubes must be room temperature, for that
take them out from refrigerator 1 hour before detection.

Table 4. The PCR program for DTlite and DTprime Thermal Cyclers
Optical
Number of Type of the
Step Temperature, °C Min. Sec. measurement
cycles step

1 94 5 00 1 Cycle
94 0 10
2 50 Cycle
62 0 20 v
5 101 … … Holding Holding
1
– holding at 25°С is allowed

Table 5. The PCR program for iCycler iQ5 thermal cyclers (with persistent well factor)
PCR/Melt Data
Cycle Repeats Step Dwell time Setpoint, °C
Acquisition
1 1
1 05:00 94.0
2 50
1 00:10 94.0
2 00:20 62.0 Real Time
3
… … 10.0 Storage

9
Table 6. The PCR program for iCycler iQ5 thermal cyclers (with dynamic factor)
PCR/Melt Data
Cycle Repeats Step Dwell time Setpoint, °C
Acquisition
dynamicwf.tmo program
1 1
1 00:30 80.0
2 05:00 94.0
2 5
1 00:20 94.0
2 00:30 62.0
3 2
1 00:20 80.0 Real Time
PCR program
4 45
1 00:10 94.0
2 00:20 62.0 Real Time
5 … … 10.0 Storage

Table 7. The PCR program for Rotor-Gene Q Thermal Cyclers

Cycling Temperature Hold Time Cycle Repeats

Cycling 80 °C 300 sec 1 time

94 °C 10 sec
Cycling 2 58 °C 5 sec 50 times
62 °C3 25 sec

Table 8. Detection channels

Specific product IC

DTprime, DTlite and IQ5 FAM HEX

Rotor-Gene Q Green Yellow

8. CONTROLS
Table 9.
Result
The controlled
Control Specific signal is Specific signal is Interpretation
step
present absent
+ + Valid
C+ PCR
- - Invalid
PCR and DNA + + Invalid
C-
extraction - - Valid
+ + Valid
PCR and DNA
IC - Valid
extraction
- Invalid

3
-take the measurement.
10
The sample is considered positive if the signal for specific DNA is present. The signal for IC could be absent
in samples with high concentration of specific DNA due to competitive priming.

The sample is considered negative if the signal for specific DNA is absent and for IC is present.

If the signal for C- is present, whole tests of current batch considered false. Decontamination required.

9. DATA ANALYSIS

In case of using DNA-Technology made Real-Time PCR Thermal Cyclers or Fluorescence Readers the
analysis performed automatically. In all other cases the analysis is based on the presence or absence of
specific signal. The controls should be also considered to exclude false positive and false negative results
(see p. 8 of the current manual). The cutoff Ct values for Rotor-Gene Q thermal cycler are 40 (specific
product) and 33 (C+). The result characterized by Ct above this value should be considered doubtful and
the whole assay should be repeated.

The analysis performed automatically.

The interpretation should be performed in accordance with table 10, 11.

Table 10. Results with HBV Conventional PCR detection Kit

Specific product (295 bp) Internal control (560 bp) Interpretation
Samples
+ Not considered Positive
- + Negative
- - uncertain
C+
+ Not considered Positive
C-
- + Negative

Table 11. Results with HBV Flash and Real-time PCR detection Kits
HBV FLASH PCR Test samples
detection Kit HBV Real-time PCR detection Kit Interpretation
Fam/Green Hex/Yellow4
“+” Cp (Ct) is specified Not considered Positive
“-“ Cp not specified
Cp (Ct) 29-34/Ct <36 Negative
(for iQ N/A)
“uncertain” Cp not specified Cp not specified
uncertain
(for iQ N/A) (for iQ5 N/A)
C+
“+” Cp (Ct) <34 Not considered Positive
C-
“-“ Cp not specified
Cp (Ct) 29-34/Ct <36 Negative
(for iQ N/A)

4
- if Cp (Ct) HEX more than specified the result is invalid!
11
 10. TROUBLESHOOTING

Table 12.
Specific signal + Specific signal - Possible cause Solution
Operation error
Repeat whole test
PCR inhibition
C+ - -
Violation of storage and
Dispose current batch
handling requirements
Dispose current batch

C- + + Contamination Perform
decontamination
procedures
IC - PCR inhibition Repeat whole test

If you face to any undescribed issues contact our representative.

11. STORAGE AND HANDLING REQUIREMENTS

Expiry date – 12 month from the date of Quality Control Department approval in compliance with all
transportation, storage and operation conditions.

All components of the HBV PCR detection Kit (except PCR-mix and C+) must be stored at temperature
from minus 18 °С to minus 22 °С over the storage period. The PCR-buffer and mineral oil can be stored
at temperatures between 2 °С and 8 °С.

The PCR-mix, C+ and PREP-NA DNA/RNA Extraction Kit must be stored at temperatures between 2 °С and
8 °С over the storage period.

Transportation can be held by all types of roofed transport with adherence to above mentioned
temperature requirements.

An expired HBV PCR detection Kit must not be used.

We strongly recommend following the instructions to get robust and reliable results.

The conformity of the HBV PCR detection Kit to the prescribed technical requirements is subject to
compliance of storage, carriage and handling conditions recommended by manufacturer.

Contact our customer service by quality issues of the HBV PCR detection Kit:

“DNA-Technology” LLC, 117587, Russia, Moscow, int. ter. Municipal District Chertanovo Severnoye,
Varshavskoye shosse, 125 Zh, building 5, floor 1, office 12

Phone: +7 (495)640.16.93,

Phone/Fax: +7 (495)640.17.71

E-mail: [email protected], www.dna-technology.ru

12
 12. SPECIFICATIONS

a. Analytical specificity: the HBV PCR detection Kit allows detection of all known HBV
subtypes. The samples containing HBV will be defined as positive. The samples
not containing HBV will be defined as negative.

b. Sensitivity: not less than 200 copies of HBV DNA per 1 mL of blood plasma.

c. Diagnostic sensivity: 99,8%.

d. Diagnostic specificity: 100%.

The claimed specifications are guaranteed when DNA extraction is performed with PREP-NA
DNA/RNA Extraction Kit.

13. QUALITY CONTROL

“DNA-Technology, Research&Production” LLC declares that. the quality control procedures performed in
accordance with ISO ISO 9001:2008 and ISO 13485:2003

14. KEY TO SYMBOLS

Caution Manufacturer

Consult instructions for use Negative control

Date of manufacture Positive control

Expiration date Cataloque number

In vitro diagnostic medical device Sufficient for

Batch code Temperature limitation

Version Upper limit of temperature

13
Annex А

Nomogram and formula for calculation of relative centrifugal force (RCF) in the speed of rotation
(RPM) depending of the rotor diameter

R1-P602-23/9EU F1-P602-21/1EU
R1-P602-S3/9EU E1-P602-50/1EU
184-8.2024.04.22
R1-P602-24/9EU E1-P602-20/1EU
F1-P602-51/1EU

14