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RTP® DNA/RNA Virus Mini Kit

Overview
Applications
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The RTP DNA/RNA Virus Mini Kit contains the pre-filled Extraction Tubes with lyophilized lysis components for viral DNA and RNA isolation from up to 200 µl of serum, plasma other cell-free body fluids, cell culture supernatants, tissue samples (max. 10 mg), blood (max. 50 µl) and rinse liquid from swabs. After lysis the samples are transferred to a spin column-based procedure with a total preparation time of PCR/ RT-PCR templates in just 20 minutes.

Starting Material

serum, urine, plasma, cell culture supernatants and other cell-free body fluids as well as swabs or tissue biopsies

Product Characteristics

Product	Starting Material	Average Yield	Preparation Time
RTP® DNA/RNA Virus Mini Kit	up to 200 μl serum, plasma, cell-free body fluids, cell culture supernatant up to 10 mg tissue samples up to 400µl rinse liquid from swabs	depending on viral titer	about 25 min

Advantages

Time saving and safer sample handling - 60 % less pipetting steps and tip consumption
One lysis tube: Ready To Prep (RTP) - pre-filled Extraction Tube with lyophilized lysis components (lysis buffer, Proteinase K), Carrier RNA and internal DNA extraction control; stable at room temperature
Universal viral nucleic acid purification system - one kit for both DNA and RNA viral purification allowing simultaneous testing of both virus types
Environment-friendly - less infectious plastic waste due to reduced hands-on steps
CE-marked in compliance with the Directive 98/79/EC on in vitro diagnostic medical devices (IVD-Directive)*

Overview

Using the RTP DNA/RNA Virus Mini Kit the sample is lysed in the pre-filled Extraction Tube containing lyophilized lysis reagents (non-chaotropic lysis buffer, Proteinase K) and carrier nucleic acid. The lysis reagents further stabilize the viral nucleic acids, inactivate RNases and DNases and enhance the selective DNA /RNA adsorption to the membrane of the SafeClick Spin Filter. Before the viral nucleic acids are eluted, the membranes are efficiently washed in order to completely remove all PCR inhibitors.
By using the pre-filled Extraction Tubes, the preparation of carrier nucleic acid solution or Proteinase K solution, as well as the pipetting of different components to the sample during the lysis, are not longer necessary. This reduces handling steps with infectious samples, processing time and contamination risk to a minimum.

*) Products which are CE-marked according to the IVD-Directive can be used for diagnostic applications in countries where this directive is recognized.

Ordering Information

Product	Package Size	Catalogue No
RTP DNA/RNA Virus Mini Kit	50 purifications	1040100200 (old: 10403002)
RTP DNA/RNA Virus Mini Kit	250 purifications	1040100300 (old: 10403003)

Process Overview

Applications

Sensitive viral RNA isolation

200 µl of human plasma samples were spiked with Influenza A virus (1:10 dilution series) and viral RNA was isolated using the RTP DNA/RNA Virus Mini Kit (blue lines) and the Invisorb Spin Virus RNA Mini Kit (red lines). The figure shows the real-time amplification results, analyzed in triplicates. The black curve represents the negative control.

Reproducible recovery of viral DNA

Viral DNA was isolated from 10 different plasma samples (200 µl) spiked with 500 HBV copies using the RTP DNA/RNA Virus Mini Kit. The viral DNA (green and blue curves) was eluted in 100 µl and 2.5 µl (12.5 copies per PCR - theoretical amount) were analyzed in a HBV specific real-time PCR assay. The mean Ct standard deviation is 0.41. The red curve represents the positive control.

Sensitivity testing of HCV virus from clinical human sample

Viral RNA was isolated from 200 µl of HCV infected patient serum sample using the RTP DNA/RNA Virus Mini Kit. The samples were analyzed using the RoboGene HCV quantitative RNA detection module. The test was performed on an ABI PRISM 7000 SDS. 5 µl of eluted HCV sample RNA was used per RT-PCR run. The analysis was performed in duplicate.

positive sample: HCV positive serum, genotype 1 2.700.000 IU per ml
negative sample HCV negative serum, obtained from healthy blood donors
The HCV positive sample was diluted in HCV negative serum 1:5 (dilution series from 500.000 IU per ml up to 30 IU per ml)

Data kindly provided by Dr. T. Köhler, Roboscreen GmbH, Leipzig, Germany.

Sensitive detection of HBV virus from clinical serum sample

Virus DNA was isolated from 200 µl HBV infected patient serum sample using the RTP DNA/RNA Virus Mini Kit and eluted in 60 µl buffer. The samples were analyzed using the RoboGene Quantification Module for Hepatitis B virus (HBV) Genomes. The test was performed on an ABI PRISM 7000 SDS. 5 µl of eluted HBV sample DNA was used per RT-PCR run.

positive sample: HBV positive serum
sample 1: 3000 genome equivalents / ml
sample 2: 5,400,000 genome equivalents / ml
negative sample: HBV negative serum, obtained from healthy blood donors

The data show the amplification of positive samples (1, 2). The other curves represent the amplification of the ready -to use HBV DNA control (dilution series from 1,000,000 up to 10 molecules per well control strip).

Data kindly provided from Dr. T. Köhler, Roboscreen GmbH, Leipzig, Germany.

Sensitive detection of the Influenza-virus from pharyngeal clinical swab samples

A dilution series of Influenza A/H3N2 virus (strain: Moskau from10⁷to 103) was prepared: The viral RNA was isolated using the RTP DNA/RNA Virus Mini Kit. 25 µl of the eluted viral RNA was used for the transcription to cDNA and 5 µl of the RT-PCR product was used for TaqMan-analysis for the positive detection of the viral infection.

Pharyngeal swab from patients with low concentration of the Influenza A virus was squeezed out in 4 ml medium. Viral RNA was isolated using the RTP DNA/RNA Virus Mini Kit from an aliquot of 200 µl of this medium. 25 µl was used for the transcription to cDNA and 5 µl of the RT-PCR product was used for TaqMan analysis for the positive detection of the viral infection.

Data kindly provided by Dr. Schweiger, Robert-Koch-Institute, Berlin, Germany