HBV DETECT ™ Real Time PCR Kit

I. Description

The HBV DETECT ™ Real Time PCR Kit is a ready-to-use kit containing all of the PCR reagents (except PCR master mix) needed for running quantitative, real-time DNA detection assays, including qPCR, in the hydrolysis probe detection format. The HBV DETECT ™ Real Time PCR Kit is designed for the in vitro quantification of DNA from HBV genotypes (A to G) in human plasma or serum from HBV-infected patients. The HBV DETECT ™ specific primers amplify an 88 base pair conserved region of the HBV X gene. The presence of RT-PCR products is measured by detection of the fluorescence emitting FAM labeled probe.

The unique features of the HBV DETECT ™ Real Time PCR Kit include high sensitivity, excellent reproducibility, wide linear range and genotype inclusivity.

Background

The World Health Organization (WHO) estimates that as of 2008, about 2 billion people worldwide are infected by HBV and about 350 million people are living with chronic infection. Furthermore, an estimated 600,000 persons die each year due to acute or chronic consequences of hepatitis B. The importance of early identification is reflected in the 25% mortality rate of adults who become chronically infected during childhood. With HBV being 50 to 100 times more infectious than HIV, and given the dire results of HBV infection, it is imperative that infection be identified. Considering the relative safety and effectiveness (95%) of the vaccine, treatment and vaccination are impeded only by the lack of identification prevalent in low and middle-income countries.

Hepatitis B is endemic in China and other parts of Asia. Most people in the region become infected with HBV during childhood. In these regions, 8% to 10% of the adult populations are chronically infected. Liver cancer caused by HBV is among the first three causes of death from cancer in men, and a major cause of cancer in women. In these areas, HBV detection and identification is difficult due to lack of accessibility available to the general populace. However, with the HBV DETECT ™ Real Time PCR Kit, a wider range of people will be able to easily attain the proper materials for identification. The HBV DETECT ™ Real Time PCR Kit is designed to assay presence of HBV DNA obtained by extraction of DNA from DBS sample.
The detection and quantification of circulatory HBV DNA provides an efficient method of diagnosing HBV infections, closely monitoring disease progression and for assessing therapeutic response.

II. Product Components and Storage Conditions

Kit Contents

This kit contains reagents for approximately one plate of 96 reactions.
HBV 10X concentrated Probe and Primer Mix (green-capped vial)
HBV positive control template dilution 1 (orange-capped vial)
Labeled tubes for preparation of HIV-1 positive control template dilution 2, 3, 4 and 5 (orange-capped vials)
HBV negative control (purple-capped vial)
HBV RNase/DNase free water (red-capped vials)

Storage and Stability

If stored at –15 to –25°C, the product is stable through the expiration date printed on the label. For short-term storage (up to 3 months) the product may be stored at +2 to +8°C. The HBV DETECT ™ Real Time PCR Kit components are light sensitive and should be kept away from the light. Avoid repeated freeze/thaw cycles and do not vortex the Probe and Primer mix.

Additional Equipment and Reagents Required

Additional equipment and reagents required to perform real-time PCR assays with Custom HBV DNA Probe and Primer mix, but not provided, include:
– DNA Extraction and Purification Kit
– Nuclease-free, aerosol-resistant tips
– Vortex mixer
– Micropipettes
– Sterile reaction tubes for preparing PCR mixes and dilutions
– Standard bench top micro centrifuge
– PCR reaction vessels (e.g., optical tubes or micro plates)
– Consumer Genetics PCR Master Mix or Applied Biosystems TaqMan® Universal PCR Master Mix
– Water, PCR grade
– Real time PCR instrument

III. Protocol

A. DNA Sample Preparation

DNA extraction kits are available from various manufacturers like QIAamp Viral DNA mini extraction kit or Bioneer Genomic DNA extraction kit. If dry blood spot (DBS) will be used for sample collection then the GSB DNA extraction and purification kit is recommended.

B. Prepare Reactions

For each 10 µl reaction, prepare the following reaction mix:
1) Thaw the solutions and, for maximum recovery of the contents, briefly spin vials in a micro centrifuge before opening.
2) In a 1.5 ml reaction tube, prepare the PCR mix for one 10 µl reaction by adding the following components:

3) Pipette 7.5 µl PCR mix into each PCR reaction vessel or well of a PCR micro plate.
4) Add 2.5 µl of template or negative control DNA. To generate a standard curve use the included positive control template in triplicate.

C. PCR

1) Following the Operator’s Manual of your instrument supplier, program the instrument with the following parameters:

2) Place tubes or micro plate in the instrument and start the reaction.
3) At the end of the reaction, follow instrument instructions for quantification/analysis.

IV. Dynamic Range of Test

Under optimal PCR conditions the HBV DETECT ™ Real Time PCR Kit have very high priming efficiencies of >95% and can detect less than 20 copies of target template

Note: The HIV & HBV PCR Kits are not available for Sale in the U.S at this moment!