HBV DNA Real-Time Quantitative in vitro Diagnostics

For use with

• Applied Biosystems: ABI PRISM® Sequence Detection System 7000, 7300, 7500, 7700
• MJ Research: Chromo 4TM Four-Color Real-Time Detector ; DNA Engine Opticon® 2
• BIO-RAD Laboratories
• iCycler iQ Real-Time PCR Detection System

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TABLE OF CONTENTS

1. INTENDED USE

2. PREFACE

3. NAME AND FUNCTION

4. PRINCIPLE OF THE TEST

5. REAGENTS PROVIDED IN THE KIT

6. STORAGE

7. ADDITIONALLY REQUIRED MATERIALS AND DEVICES.

8. GENERAL PRECAUTIONS FOR USERS   

9. SPECIMEN PREPARATION AND STORAGE.

10. PREPARING THE PCR

11. DATA ANALYSIS

12. PERFORMANCE VALIDATION

13. TROUBLESHOOTING

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1.  Intended use

HBV RTQ is an in vitro diagnostic kit, which quantifies hepatitis B viral load in human serum.

2. Preface

Hepatitis B is a serious disease caused by the hepatitis B virus that attacks the liver. It
can cause lifelong infection, cirrhosis (scarring) of the liver, liver cancer, liver failure, and death, and has become a significant problem of public health management. Almost one in every ten adults that has been infected by HBV develops some forms of chronic liver diseases and becomes a long-term carrier of HBV. As a result, screening of Hepatitis B infection is urgently needed.

Serological markers such as HBsAg and HBeAg are commonly used as diagnostic indicators of acute or chronic HBV infection. However, owing to the strain variance,
the use of these markers monitoring HBV disease progression is limited, and directly
quantifying HBV DNA in serum or plasma provides a better way for viral infection
and replication measurement.

3. Name and function

HBV_RTQ Assay is a molecular diagnostic kit. It is for the use of HBV screening and quantitative test in human serum or plasma, and plays an important role in anti-viral drug efficacy evaluation.

4. Principle of the test

The kit is intended for the in vitro detection and quantification of Hepatitis B virus DNA in human serum or plasma utilizing Polymerase Chain Reaction (PCR) and TaqMan probe technology. The HBV Pm and HBV Pb contain primer pairs and TaqMan probes specific for HBV target/standard and internal control. The HBV DNA is detected in the FAM channel, and the internal control is detected in the VIC channel.

The HBV Standard is a non-infectious DNA molecule containing the identical primer
and probe binding sites the same as the HBV target. HBV viral load is calculated by
interpolation method utilizing the External Standard Curve, which is generated by
HBV Standards (QS1~QS5) in every run.

The internal control (IC), which is a special designed non-infectious DNA molecule, is incorporated into each standard and specimen sample and carried through all
amplification and detection steps. It works as an indicator for PCR performance.

5. Reagents provided in the kit

Labeling and contents Volume  (µl)      Quantity 4HBPA2 48 tests           Storage

HBV Master                      600 µl                     1                                   2 ~ 8°C
HBV Pm                             48 µl                     1                              -15 ~ -25°C
HBV Pb                              24 µl                     1
HBV IC                               48 µl                     1
H2O                                1000 µl                     1
HBV QS 1 (1×1010 cop/ml)  50 µl                     1
HBV QS 2 (1×108 cop/ml)    50 µl                     1
HBV QS 3 (1×106 cop/ml)    50 µl                     1
HBV QS 4 (1×105 cop/ml)    50 µl                     1
HBV QS 5 (1×104 cop/ml)    50 µl                     1

QS = Quantitation standard
IC = Internal Control

6. Storage

The HBV Master should be stored at 2-8°C and other kit components should be stored
at -20°C. These kit components are stable until expiry date stated on the label. Repeated
thawing and freezing should be avoided.

 

7. Additionally Required Materials and Devices

• Latex gloves, powderless
• Microcentrifuge
• 1.5 ml DNase-free centrifuge tubes
• 10µl, 20µl, 200µl micropipettes and disposable DNase-free tips with aerosol barrier
• Viral DNA Extraction Kit (QIAamp DNA Blood Mini Kit, Cat. No. 51106, or Roche High Pure Viral Nucleic Acid Kit, Cat.No.1858874, are recommended)
• ABI PRISM® Sequence Detection System 7000/7300/7500/7700, ABI 96-well reaction plates with optical adhesive covers (Cat. No. 4311971) or optical tubes (Cat. No. N801-0933) and optical caps (Cat. No. 4323032)
• MJ Chromo 4TM Four-Color Real-Time Detector, DNA Engine Opticon® 2, MJ research 0.2 ml low-profile strip tubes Cat. No. TLS-0801 natural color or Cat. No. TLS-0851 white color.
• BioRad iCycler iQ Real-Time PCR Detection System

8. General Precautions for users

This reagent kit is for in vitro diagnosis only.This reagent must not be used after expiration date.

• Do not pool reagents from different lots or from different bottles of the same lot.
• Reagents must be protected form microbial contamination.
• Positive control is made of selected partial sequence from HBV. For safety, it must be treated as infectious material.
• Thaw all components thoroughly at room temperature, mix the components and centrifuge briefly before starting an assay.
• Do not smoke, eat or drink in work areas, or pipet by mouth.
• Latex gloves must be worn when handling reagents or specimens and must be changed before leaving the area. Wash hands thoroughly afterwards.
• To avoid cross contamination between specimens and reagents, workflow in the laboratory must proceed in a uni-directional manner from the Pre-Amplification Area to Post-Amplification Area.
• Thoroughly clean and disinfect all work surfaces with 5% sodium hypochlorite.
• Before disposal, all waste materials should be properly disinfected by autoclave for at least one hour at 121.1°C or incineration.
• To performing dual-color detections with both FAM-labeled and VIC-labeled TaqMan Probes in a single tube, color calibration must be done to avoid the fluorescence residual crosstalk between channels.

9. Specimen preparation and storage

1 Sample collection

The kit is for use with serum or plasma specimens only. Serum may be collected in a
Serum Separator tube. Whole blood should be collected into sterile EDTA or CITRATE blood collection tubes. Specimens anti-coagulated with heparin are unsuitable for this test. With 6 hours of blood draw, blood collection tubes must be centrifuged at 3000 rpm for 10 minutes at room temperature to collect serum or plasma. Transfer serum or plasma to sterile polypropylene tube and store at -20°C. It is recommended that specimens be store in 250µl aliquots in sterile, 2ml polypropylene screw cap tubes.

2 Sample Transport

Transportation of whole blood, serum or plasma must be complied with country, federal, state and local regulations for the transport of etiologic agents. Whole blood must be transported at 2-25°C and processed within 6 hours of collection. Serum and plasma may be transported at 2-8°C or frozen.

3.DNA isolation

Purify target DNA from 200µl sample serum or plasma by using commercial viral
DNA extraction kit, such as QIAamp DNA Blood Mini Kit or Roche High Pure Viral
Nucleic Acid Kit. Please carry out the DNA extraction according the manufacturer’s
instructions. Elute the HBV viral DNA by adding 200µl DNase-free H2O. If HBV DNA is eluted by adding 50µl DNase-free H2O, then the DNA will be 4x concentrated. The analysis results should divide 4 to get the correct DNA concentration result.

IMPROTANT: The QS1-5 are non-infectious HBV DNA and ready to use in PCR standard curve generation. DO NOT processing DNA extraction towards QS1-5.

10. Preparing the PCR

In every PCR run, please make sure that quantitative standards and one negative control (water, PCR grade) are included. All reagents need to be thawed thoroughly and centrifuged briefly before use. For the preparation of the PCR assay please use the following pipetting scheme.

1. Mix HBV Master, HBV Pm, HBV Pb and HBV IC well, then centrifuge briefly. Dispense 15µl reagent mix to the optical tube (or plate).

2. Add 10µl QS1, QS2, QS3, QS4 and QS5 to optical tubes as standards. Mix by pipetting gently.

3. Add 10µl H2O to one optical tubes as HBV negative controls. Mix by pipetting gently.

4. Add 10µl DNA isolated from samples to optical tubes. Mix by pipetting gently. Seal the tubes(plate) and centrifuge briefly. Store at 4°C until the real-time PCR machine is programmed and ready to use.

Component     Volume (µl) 

HBV Master         12.5
HBV Pm                1.0
HBV Pb                 0.5
HBV IC                  1.0
Sample/ QS/ H2O 10.0

Total                     25.0

Programming

The program runs approximately for 2 hours and 10 minutes.

Times and Temperatures

Initial Steps                              Each of 45 Cycles

                                              Melt Anneal    /   Extend

2 min 50°C/10 min95°C            15 sec95°C       1 min58°C

HBV Standards (QS1-5), unknown samples, and no template controls dye settings:

.. Fluorescence detection stage
..Anneal/Extend stage (some machine can choose this item)
.. Detection of HBV DNA
.. Reporter dye “FAM”
.. Quencher dye “TAMRA” (some machine can choose this item)
.. Detection of HBV Internal Control
.. Reporter dye “VIC”
.. Quencher dye “TAMRA” (some machine can choose this item)

11. DATA Analysis

1. A signal is detected in the FAM channel

.. The sample contains HBV DNA. The analysis software calculates HBV DNA
concentrations. If the HBV DNA is concentrated or diluted during the isolation step, the HBV DNA actual concentration needs to be recalculated by considering the extraction factor.

.. Signal from VIC channel is not critical. The amplification of the internal control can be inhibited by competition from the positive HBV PCR.

2. No signal from the FAM channel, but a signal appears in the VIC channel.
.. No HBV DNA is detectable. It can be considered negative.

3. No signal in either the FAM channel or VIC channel.
.. No diagnosis can be concluded.  

12. Performance validation

The performance of each PCR run can be validated by the information generated by standard curve and NTC. The standard curve and NTC should be qualified by following criteria:

Items                         Criteria 

Ct of QS1                 12 = Ct = 16

Ct of QS2                 18 = Ct = 22

Ct of QS3                 24 = Ct = 28

Ct of QS4                28 = Ct = 32

Ct of QS5                32 = Ct = 36

R2 of standard curve1 R2 = 0.90

Slope of standard curve2   -4.35 = Slope = -2.75

IC of NTC3,4                       +

1. The linearity of five external standards detected by PCR system must be larger than 0.90 (R2 = 0.90).
2. The slope represents the overall reaction efficiency. To achieve efficiency for the standard curve between 1.7 (85%) and 2.3 (115%), the slope has to be between –4.35 and –2.75
3. Negative sample or NTC. FAM channel Ct value is undetectable and VIC channel Ct should be detectable.
4. Positive sample. VIC channel Ct may or may not be detected.
5. The sensitivity of the kit is 1000 copies/ml serum.  

13. Troubleshooting

1. No signal or weak signal in the FAM channel from the Standard (HBV QS 1-5).

.. Incorrect programming of the ABI SDS Instrument.
.. Repeat the PCR with corrected settings.
.. The reagents have been thawed and frozen too often, or stored improperly.
.. Store the reagents at -15 ~ -25°C
.. Avoid repeated thawing and freezing.
.. Prevent HBV Pb from light.

2. Weak or no signal of the Internal Control in the VIC channel, and no signal in the FAM channel.

.. The HBV IC has been thawed and frozen too often. IC DNA degradation
.. Use new HBV IC
.. The PCR was inhibited.
.. Make sure that you use a recommended isolation method and stick closely to the manufacturer’s instructions.
.. Dilute the specimen 10 folds and detect it again.

3. Log-linear phase of amplification just starts when the amplification program finishes.

.. Very low starting amounts of nucleic acids.
.. Use concentrated starting material.

4. Fluorescence intensity varies

.. Air bubble is trapped in the optical tube.
.. Before PCR reaction, centrifuge the optical tube (plate) at 1000rpm for 2 min.
.. Pipetting errors
.. Repeat run if needed.
.. Skin oil on the surface of the optical cap.
.. Always wear gloves when handling.

5. Negative control sample is positive

.. Contamination
.. Exchange all critical solutions.
.. Pipet on a clean bench.