The Roboscreen HBV Kit comes with all the reagents necessary to perform all steps from viral extraction through detection via real-time quantitative PCR (qPCR). The kit also includes standards and amplification controls. The viral extraction kit extracts DNA and RNA, in the same tube, from liquid samples like blood and serum. For extraction of nucleic acid from Hepatitis B Virus (HBV), we use 1 ml of serum as the starting material. The extraction of viral DNA is column-based which makes for a simple extraction with good yields. The extraction process includes the following steps: Before setting up the reactions, 20 ml of ethanol must be added to Washing Solution I and 48 ml of ethanol must be added to Washing Solution II. The first step in the extraction is a precipitation step in which we combine our sample with the precipitation buffer. Precipitation is followed by a lysing step in which we add resuspension buffer and incubate for 30 minutes at 65ºC. Next, we bind the liberated DNA with binding solution and place a spin filter in a clean tube. We then transfer the sample into the filter and centrifuge at 10000 rpm. This binding is followed by two washing steps and, finally, we elute our DNA in the elution solution which is provided in kit.
Roboscreen amplification kits are TaqMan®-based assays. The amplification module includes DNA tubes which contain 8 different amounts of HBV control DNA with amplification enhancers. Sample tubes are also coated with amplification enhancers. The HBV Reagent Mix is a mixture of lyophilized primers and probes. MgCl2, 10X PCR buffer, BSA solution and PCR grade water are also provided. The total reaction volume is 25 uL out of which 5 uL is from our DNA sample and the remaining 20 uL is made up of: 10.7 uL PCR grade water, 2.5 uL 10X PCR buffer, 1.5 uL 2 (3 mM), 5 uL 5X HBV Reagent Mix, 0.3 uL (1.5 U) of AmpliTaq Gold® which is provided with the kit. The thermal cycle program to run the HBV assay is: Hold for 10 minutes at 95ºC followed by 50 cycles of 95ºC for 15 seconds and 59ºC for 30 seconds. The total reaction time is 1 hour 5 minutes.
The Roboscreen extraction protocol is very user friendly. Since there is no requirement for repetitive centrifugation and continuous incubation, we can extract DNA from our starting sample in just 45 minutes. As far as the amplification module is concerned, we are happy with the reproducible results this kit generates. The kit also has a broad range of detection; the standards provided range from 1000000 to 10 molecules per tube. But I personally feel there are also a few limitations with this module: A lot of pipetting is required during the master mix preparation. Also, many of the required solutions are in separate tubes so the chances of error increases. The other limitation of this kit is the shelf life of the reagent mix, one reagent mix tube is viable for 2-3 weeks.
I would recommend this product to anyone who wishes to go straight through from nucleic acid extraction to real-time qPCR.
Tarun Jain
Product Specialist
Biotron
Molecular Diagnostics