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Contents 1 2 3 3 4 6 8 9 10 11 13 15 16 17 18 Introduction RTP® technology - Ready To Prep Optimized protocols Implementation on robotic workstations Product selection guide Walk-away DNA/RNA purification on the InviGenius® STRATEC Molecular Virus kits - spin columns Optimal viral RNA/DNA purification Simultaneous viral RNA/ DNA purification Invisorb® Virus HTS kits - 96 well InviMag® Virus kits - magnetic beads Simultaneous viral RNA/ DNA purification Detection of internal controls References About STRATEC Molecular Contact information STRATEC Molecular GmbH Robert-Rössle-Str. 10 D-13125 Berlin Germany Phone: +49-30-9489 2901/2910 Fax: +49-30-9489 3795 www.invitek.de 6I7j06/04/2011 Smarter Nucleic Acid Sample Preparation for Virus Applications Patented complete solutions for customer applications Sample preparation kits for the isolation of viral RNA and/or DNA from: whole blood, serum, plasma, cell-free body fluids, swabs stool samples tissue samples cell culture supernatants Introduction The STRATEC Molecular Virus Kits provide rapid and efficient purification of viral RNA and DNA from a variety of starting materials. The RTP® product range enables a tremendous simplification for DNA/RNA purification from pathogen containing samples. o efficient recovery of high-quality viral RNA/DNA allowing for very sensitive virus detection o linear yields - efficient purification over a wide range of viral titers o various isolation systems available to suit your special needs o RTP® technology: the most convenient processing for pathogen-containing samples The STRATEC Molecular Virus Kits are available in single sample format, in 96 well format using filter plates for use on robotic workstations, on a vacuum manifold or centrifuges. Furthermore STRATEC Molecular offers the magnetic bead based InviMag® Virus Kits in manual single sample format or for automated use on the InviGenius® or other magnetic workstations like the KingFisher® machines from ThermoFisher Scientific. All STRATEC Molecular Virus kits are CE-marked in compliance with EU Directive 98/79/EC in Europe* and are supplied with exact performance specifications, assuring highly reliable nucleic acid purification. For safe handling and a reduced danger of contamination or infection capped spin filters are supplied in all pathogen spin kits. *) USA: for research use only - not for use in diagnostic procedures 1 RTP® technology – Ready To Prep The RTP® product range is based on STRATEC Molecular´s unique non-chaotropic chemistry. Everything required for DNA/RNA sample preparation - lysis buffer, Proteinase K, carrier RNA for enhanced recovery of low amounts of target sequences, and internal DNA extraction control (for monitoring extraction efficiency and PCR amplification) is provided in a single Extraction Tube – lyophilized, ready-to-use & stable at room temperature! Advantages: o Improve Efficiency – Time saving and safer sample handling 60 % less pipetting steps and tip consumption o Improve Efficiency - One lysis tube pre-filled Extraction Tube with lyophilized lysis components; stable at room temperature o Maximize Capabilities - Universal viral nucleic acid purification system one kit for both DNA and RNA viral purification allowing simultaneous testing of both virus types o Improve Safety - Environment-friendly less infectious plastic waste due to reduced hands-on steps 2 Optimized protocols Invisorb® isolation technology The sample is lysed in an optimized lysis buffer containing Carrier RNA and proteins are degraded during the lysis with Proteinase K. The liberated RNA or DNA is bound onto the membrane of the capped RTA Spin Filter. Contaminants are removed by repeated washing steps and the purified viral nucleic acid is finally eluted. RTP® isolation technology Using the RTP® technology the sample is lysed in the pre-filled Extraction Tubes with lyophilized lysis components. 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 spin column. 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. As a result of high extraction efficiency, the kits realize high virus detection sensitivities. Implementation on robotic workstations With its innovative design, the InviGenius® from STRATEC Molecular streamlines the sample preparation workflow providing true sample-“in”, high quality and ready to use DNA or RNA-“out” technology. It delivers exceptional process safety and a completely monitored purification process for viral nucleic acids. The adaptation of well established STRATEC Molecular HTS kits to common liquid handling workstations, e.g. the Eppendorf epMotion® 5075 platforms or the Tecan workstations allows for a simple and automated isolation of viral nucleic acids. Manual handling of potentially infectious samples is minimized, ensuring safety to the user and reliable processing of samples. The procedures are designed to avoid sample-to-sample cross contamination. The InviMag® Virus Kits for medium and high throughput applications using magnetic particles are designed for use on the KingFisher mL, KingFisher 96 and KingFisher Flex platforms. o special customer tailored solutions are also available on request o programs can simply be downloaded from www.invitek.de o for adaptation to robotic platforms please contact STRATEC Molecular: +49 30 9489 2901/2910 or [email protected] 3 Product selection guide Format Starting material for isolation of viral DNA and/or RNA Product name up to 200 µl serum, plasma, cell-free body fluids, cell culture supernatant; up to 10 ® RTP DNA/RNA Virus Mini Kit mg tissue samples; up to 400 µl rinse liquid from swabs Single tube, membrane adsorption based Article number Package size 1040100200 1040100300 50 preps 250 preps 1040400200 1040400300 50 preps 250 preps up to 200 µl serum, plasma, cell-free body fluids, cell culture supernatants; 6 up to 50 µl whole blood; 1 x 10 ® Invisorb Spin Virus RNA Mini Kit mammalian cells; max. 20 mg tissue sample; max. 50 mg stool sample; rinse liquid from swabs 1040300200 1040300300 50 preps 250 preps up to 200 µl plasma, serum, whole blood, cell-free body fluids, rinse liquid from swabs 1040200200 1040200300 50 preps 250 preps 1 – 5 ml pooled serum or plasma ® RTP DNA/RNA Virus Supersense Kit ® Invisorb Spin Virus DNA Mini Kit ® Invisorb Virus RNA HTS 96 Kit/ C (for use on a centrifuge) ® Invisorb Virus RNA HTS 96 Kit/ V up to 200 µl serum, plasma, cell-free body (using a vacuum manifold) fluids, rinse liquid from swabs; max. 50 ® mg stool samples Invisorb Virus RNA HTS 96 Kit/ ep (for use on epMotion® platforms) HTS – 96 well ® Invisorb Virus RNA HTS 96 Kit/ X automated, centrifuge, vacuum membrane adsorption based (for use on X-tractor Gene™) ® Invisorb Virus DNA HTS 96 Kit/ C (for use on a centrifuge) ® Invisorb Virus DNA HTS 96 Kit/ V up to 200 µl serum or plasma cell-free body fluids, whole blood, rinse liquid from swabs (using a vacuum manifold) ® Invisorb Virus DNA HTS 96 Kit/ ep (for use on epMotion® platforms) ® Invisorb Virus DNA HTS 96 Kit/ X (for use on X-tractor Gene™) up to 200 µl serum, plasma, cell-free body ® fluids, cell culture supernatant InviMag Virus DNA/RNA Mini Kit up to 10 mg tissue samples (for manual use on the InviMag® Rack) up to 400 µl rinse liquid from swabs Low throughput, up to 200 µ plasma, serum, cell-free body magnetic ® fluids, rinse liquid from swabs InviMag Virus RNA Mini Kit bead based up to 50 mg stool samples (for manual use on the InviMag® Rack) up to 10 mg tissue samples up to 200 µl plasma, serum, whole blood, cell-free body fluids, rinse liquid from swabs ® InviMag Virus DNA Mini Kit (for manual use on the InviMag® Rack) 4 7043300200 7043300300 7043300400 7043310200 7043310300 7043310400 2x96 preps 4x96 preps 7143320200 24x96 preps 7143320300 7143320400 7143310200 7143310300 7143310400 7042300200 7042300300 7042300400 7042310200 7042310300 7042310400 2x96 preps 4x96 preps 24x96 preps 7142320200 7142320300 7142320400 7142310200 7142310300 7142310400 1440100200 1440100300 50 preps 250 preps 1440300200 1440300300 50 preps 250 preps 1440200200 1440200300 50 preps 250 preps Product selection guide Format Starting material for isolation of viral DNA and/or RNA Article number Package size up to 200 µl serum, plasma, cell-free body ® InviMag Virus DNA/RNA Mini Kit/ fluids, cell culture supernatant KFmL up to 10 mg tissue samples ® (automated, for use on KingFisher mL) up to 400 µl rinse liquid from swabs 2441150100 2441150200 2441150300 2441150400 15 preps 75 preps 150 preps 300 preps up to 200 µl plasma, serum, cell-free body ® fluids, rinse liquid from swabs InviMag Virus RNA Mini Kit/ KFmL ® (automated, for use on KingFisher mL) up to 50 mg stool samples up to 10 mg tissue samples 2443110100 2443110200 2443110300 2443110400 15 preps 75 preps 150 preps 300 preps Medium up to 1 ml plasma, serum, cell-free body throughput fluids, rinse liquid from swabs automated, magnetic bead based Product name 200 µl serum, plasma, cell-free body fluids, rinse liquids from swabs, 50 mg stool sample, 10 mg tissue sample 7443710100 1x24 preps ® InviMag Virus RNA Midi Kit/ KFflex24 7443710200 5x24 preps (automated, for use on KingFisher® Flex) 7443710300 10x24 preps ® InviMag Virus RNA Mini Kit/ IG (automated, for use on InviGenius®) up to 200 µl plasma, serum, whole blood, ® InviMag Virus DNA Mini Kit/ KFmL cell-free body fluids, rinse liquid from (automated, for use on KingFisher® mL) swabs 2443120100 8x12 preps 2443120200 16x12 preps 2443120300 32x12 preps 2442110100 2442110200 15 preps 75 preps up to 1 ml of plasma, serum, whole blood, 7442710100 1x24 preps ® InviMag Virus DNA Midi Kit/ KFflex24 7442710200 cell-free body fluids, rinsed liquid from 5x24 preps ® swabs, cell-free cell culture supernatants (automated, for use on KingFisher ml) 7442710300 10x24 preps 200 µl whole human blood, serum, plasma, cell-free body fluids, rinse liquid from swabs ® InviMag Virus DNA Mini Kit/ IG ® (automated, for use on InviGenius ) 2442120100 8x12 preps 2442120200 16x12 preps 2442120300 32x12 preps up to 200 µ plasma, serum, cell-free body ® InviMag Virus RNA Mini Kit/ KF96 fluids, rinse liquid from swabs (automated, for use on KingFisher® 96 and up to 50 mg stool samples KingFisher® Flex) up to 10 mg tissue samples 7443300100 7443300200 1x96 preps 5x96 preps HTS – 96 well up to 200 µl plasma, serum, whole blood, InviMag® Virus DNA Mini Kit/ KF96 automated, (automated, for use on KingFisher® 96 and cell free body fluids, rinse liquid from KingFisher® Flex) magnetic swabs 7442300100 7442300200 1x96 preps 5x96 preps up to 200 µl serum, plasma, cell-free body InviMag® Virus DNA/RNA Mini Kit/ fluids, cell culture supernatant, rinse liquid KF96 (automated, for use on KingFisher® 96 and from swabs KingFisher® Flex) up to 50 µl blood up to 10 mg tissue 7441050100 7441050200 1x96 preps 5x96 preps bead based ® For simultaneous nucleic acid purification from bacteria and viruses the RTP Pathogen Kit (1040500200) is also available. 5 Walk-away DNA/RNA purification on the InviGenius® The InviGenius® provides advanced and reliable performance in automated nucleic acid sample preparation. The use of this system in combination with ® the well established magnetic bead based InviMag technology for DNA and RNA purification will allow for increased standardization and streamlining of laboratory workflows. Innovative functions and optimized protocols enable reliable results for high quality DNA/RNA purification. The InviGenius® automated system, with built-in touch screen and computer, provides efficient automation of the sample preparation workflow. Principle The InviGenius® controls an array of magnetic rods that can attract or release magnetic particles. After sample lysis the InviGenius® transfers magnetic particles with bound nucleic acids through the buffers – unlike other systems – and elutes pure nucleic acids ready-to-use for subsequent downstream analysis. Advantages o o o o o o o purify DNA/RNA from up to 12 samples proven performance-leading magnetic-particle chemistry best in error prevention advanced process safety and standardized sample preparation highest degree of automation total in-process control data storage, backup and archiving Sample dilution Mean Ct value InviGenius plasma 1 31.89 InviGenius plasma 2 31.92 InviGenius plasma 3 31.88 InviGenius plasma 4 31.90 InviGenius plasma 5 31.85 InviGenius plasma 6 31.86 Positive Control 30.30 Negative Control Fig. 1 Fully automated isolation of viral RNA from Influenza A 6 different plasma samples (200 µl) were spiked with 1 µl from the same lot of a standard Influenza A stock. The viral RNA was isolated using the InviMag® Virus RNA Mini Kit/ IG and was eluted in 200 µl Elution Buffer. 2.5 µl of the eluted RNA were analyzed with a realtime RT-PCR using a commercial Influenza A detection kit. 6 Walk-away DNA/RNA purification on the InviGenius® Fig. 2 Linear range of viral DNA isolation efficiency Viral DNA from a series of different HBV concentrations (50 copies/ prep – 250.0000 copies/ prep) in spiked plasma samples were isolated using the InviMag® Virus DNA Mini Kit/ IG. 200 µl starting material was used for viral DNA extraction. The eluates were analyzed by a probe based, HBV specific real-time PCR. The figure shows the mean Ct values for 5 different concentrations of HBV in plasma. The corresponding Ct values for the real-time PCR are listed in the table. Each test was performed in triplicates. HBV copies per prep HBV copies per PCR mean Ct value Std. deviation 50 1.21 35.75 0.64 500 12.5 33.28 0.09 5000 125 30.74 0.07 50.000 1250 27.50 0.13 250.000 6250 25.39 0.23 7 STRATEC Molecular Virus kits - spin columns Invisorb® technology Lysis of starting material RTP® technology RTP® Supersense technology Lysis of starting material in the Extraction Tube Precipitation Adjust binding conditions Adjustment of binding conditions Resuspension of the pellet Binding of viral nucleic acids Lysis of starting material in the Extraction Tube Binding of viral nucleic acids Washing Removal of ethanol Adjust binding conditions Binding of viral nucleic acids Washing Removal of ethanol Elution of viral DNA and/or RNA Washing Removal of ethanol Elution of viral DNA or RNA Elution of viral DNA and/or RNA 8 Optimal viral RNA/DNA purification Use the Invisorb® Spin Virus RNA Mini Kit for reliable isolation of high-quality RNA from viruses found in a diverse range of starting materials. The kit simplifies viral RNA isolation by combining efficient lysis of the starting material and the inactivation of exogenous and endogenous RNases to prevent degradation. Fig. 3 Sensitive detection of Norwalk Virus from stool samples positive control Viral RNA was isolated from 50 mg NLV infected fecal sample using the Invisorb® Spin Virus RNA Mini Kit. The extracted viral RNA was amplified on a RotorGene™ 3000 using a NLV specific PCR. 10 µl of eluted NLV sample RNA was used per cDNA synthesis. Norwalk virus infected patient sample 2 Norwalk virus infected patient sample 1 negative control The Invisorb® Spin Virus DNA Mini Kit is an effective solution for isolation of high quality viral DNA from viruses found in a diverse range of starting materials. Recoveries permit detection of less than 250 copies/ml of viral DNA by qPCR (inhouse), shows that the Invisorb® Spin Virus DNA Mini Kit provides 100% detection rate of HBV DNA from a 250-copy/ml (5 copies per PCR). Fig. 4 Linear yields and reproducibility Different viral DNA extractions were performed from HBV spiked plasma with a dilution series (50 copies/ prep – 50000 copies/ prep) using the Invisorb® Spin Virus DNA Mini Kit. Standard deviations were determined for HBV dilution series in the linear range of the appropriate downstream assay. For precise analysis, the same downstream assay was used as was for the determination of the linear range (preparation from given copy numbers was used as a standard). 9 Simultaneous viral RNA/ DNA purification 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 (see figure), other cellfree body fluids, cell culture supernatants, tissue samples (max. 10 mg) and swabs. After lysis the samples are transferred to a spin column-based procedure with a total preparation time of PCR/ RTPCR templates in just 20 minutes. Fig. 5 Reproducible recovery of viral DNA for samples with low virus load 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. The RTP® DNA/RNA Virus Supersense Kit is the ideal tool for the concentration and simultaneous viral DNA/RNA isolation from max. 5 ml pooled serum, plasma or other liquid sample in about 60 min. The kit combines a method to concentrate viral nucleic acids, followed by nucleic acid purification using the Extraction Tube. The procedure provides increased sensitivity in viral-load monitoring and other applications where high viral nucleic acid recovery is essential. (A) saturation curve Fig. 6 (B) reverence plot Quantification of Hepatitis C virus genomes Quantification of HCV genomes using a Rotor-Gene™ 3000 instrument. A HVC-RNA positive human serum sample with 677,00 lU/ml (quantification was performed using the Roche Amplicor HCV assay) was diluted in HCV negative human serum and viral RNA isolated with the RTP DNA/RNA Virus Supersense Kit (sample Volumen:1ml, elution volume: 60 µl). 5 µl of the eluate was used for each RT-PCR. (A) The saturation curves show the amplification of the HCV-RNA positive samples with following concentration: red curves 67,700lU/ml; green curves 6,770 lU/ml, pink curves 677 lU/ml; black curves 68lU/ml. The blue curves represent the amplification of the HCV Control cRNA (standard, Roboscreen). The HCR-RNA negative sample (NS) and the non template control (NTC) did not show any amplification. (B) Reference curve of HCV Control cRNA (Roboscreen). The reaction efficiency of this experiment is 0.98, and the R-value is 0.997. (Data kindly provided by Dr. Rost, Roboscreen GmbH) 10 Invisorb® Virus HTS kits – 96 well (membrane adsorption) Procedure Lysis of starting material Adjustment of binding conditions Binding of viral RNA or DNA The Invisorb® Virus HTS 96 Kits are designed for simultaneous processing of 96 samples. Viral RNA or DNA purification can be carried out on a centrifuge, as a vacuum driven process on a robotic workstation for automated isolation, or on the Invisorb® 96 Vacuum Manifold. The sample is lysed in an optimized lysis buffer containing carrier nucleic acids. The addition of Carrier RNA (provided with the kits) is necessary for the enhancement of RNA or DNA recovery in samples with low virus load. Carrier RNA also stabilizes nucleic acids in samples with very small nucleic acid concentrations. The viral proteins are degraded during the lysis with Proteinase K. The viral nucleic acids bind to the filter membrane, followed by washing steps and the final elution. In combination with a robotic workstation e.g. the epMotion® 5075 VAC, the Invisorb® Virus HTS 96 Kits enable automated purification of viral RNA or DNA from 1 – 96 samples in less than 2 hours with no hands-on time during the run. Washing steps Elution of viral RNA or DNA Fig. 7 Virus materials RNA extraction from different starting A test using urine, rinse fluid from cervix swabs from different patients and plasma as control was performed using the Invisorb® Virus RNA HTS 96 Kit/ ep on the epMotion® 5075 VAC platform. All samples were spiked with the same lot of Influenza A virus with a dilution of 1:200. The figure shows that the extraction efficiency is independent from the matrix. Sample matrix 11 Mean Ct value Std. deviation urine 28.26 0.27 plasma 28.14 0.69 rinse liquid from cervix swab 27.89 0.45 Invisorb® Virus HTS kits – 96 well Sample dilution 3 dilution 2 dilution 1 Fig. 8 Mean Ct value Std. deviation CMV plasma dilution 1 30.75 0.11 CMV plasma dilution 2 34.63 0.56 CMV plasma dilution 3 37.02 0.20 CMV plasma dilution 1 30.83 CMV plasma dilution 2 35.03 CMV plasma dilution 3 36.88 Reproducible and sensitive isolation of viral DNA from HBV spiked samples Different viral DNA extractions were performed on the epMotion® 5075 VAC platform from a dilution series of CMV spiked plasma using the Invisorb® Virus DNA HTS 96 Kit/ ep. From a 1:10.000 dilution four further dilutions of 1:10 (10-5 – 10-8) were made and extracted from plasma samples. The figure shows the amplification results for 3 different concentrations of CMV in plasma. The corresponding Ct values and standard deviations for the real-time PCR are listed below. 12 InviMag® Virus kits - magnetic beads Procedure semi-automated nucleic acid isolation manual sample preparation manual nucleic acid isolation Lysis of starting material (in the Extraction Tube) Addition of InviMag® beads and Binding Solution to the lysate Lysis of starting material (in the Extraction Tube) Addition of InviMag® beads and Binding Solution to the lysate Viral nucleic acids bind to the magnetic particles Viral nucleic acids bind to the magnetic particles automated nucleic acid purification on the KingFisher platforms Magnetic separation Washing of the particle fixed nucleic acids Magnetic bead separation Elution of viral nucleic acids Magnetic separation Magnetic separation Washing of the particle fixed viral nucleic acids Magnetic separation Elution of viral nucleic acids Magnetic separation Pure viral nucleic acids Pure isolated viral nucleic acids 13 InviMag® Virus kits - magnetic beads The InviMag® Virus Kits allow for rapid and economical purification of viral RNA and/or DNA from up to 200 µl (mini) or 1 ml (midi) of virus containing samples using magnetic beads. The InviMag® Virus Mini Kits have been designed for manual use on the InviMag® Rack. The InviMag® Virus Kits/ KF have been designed for automated preparation on the KingFisher workstations. Beside the pre-filling of all cavities, all processing steps are performed by the robot. The manual kit follows the same principle. The sample is lysed in an optimized lysis buffer containing Proteinase K and Carrier RNA. The lysis step can be carried out direct on the platform at ambient temperature or outside on a thermomixer at 56°C while continuously shaking. In this case, the lysed samples are transferred onto the workstation to the subsequent automatic purification procedure based on magnetic beads. Sample Fig. 9 mean Ct value Std. deviation Urine 32.23 0.23 Plasma 1 32.17 0.13 Plasma 2 32.18 0.37 Positive Control 31.94 0.45 Reproducible recovery of viral RNA from diverse biological samples Viral RNA was isolated using the InviMag® Virus RNA Mini Kit/ KF96 from urine and different plasma samples. All samples (urine, plasma) were spiked with same lot of Influenza A virus with a dilution of 1:200. 200 µl starting material was used for the viral RNA extraction of 2.5 µl of the eluted RNA and was analyzed as triplett using an Influenza A specific RT-PCR. Sample mean Ct value Std. deviation Cervix Swab 37.35 0.74 Plasma 36.56 0.63 Blood 37.80 0.27 Positive control 31.16 0.09 Fig. 10 Reproducible recovery of viral DNA from diverse biological samples Viral DNA was isolated using the InviMag® Virus DNA Mini Kit/ KF96 from rinse liquid from cervix swab, plasma and whole blood. All samples were spiked with the same lot of HBV (500 copies/ prep). 200 µl starting material was used for viral DNA extraction. 5 µl of the eluted DNA was analyzed as triplett using a HBV specific PCR. 14 Simultaneous viral RNA/ DNA purification The InviMag® Virus DNA/RNA Kits contain the pre-filled Extraction Tubes with lyophilized lysis components for simultaneous isolation of high quality viral DNA and RNA from a diverse range of clinical samples using magnetic beads. Fig. 11 Isolation and detection of BVD-Virus from plasma pools RNA from bovine diarrhea virus from cattle was isolated from 5 ml pooled plasma sample (5 ml) with the InviMag® Virus DNA/RNA Kit/ KFmL on the KingFisher mL platform. Only 100 µl of plasma sample per animal of maximum 50 animals was used to produce the 5 ml pools. From each pool an aliquot of only 200 µl serum or plasma was used for viral RNA isolation. Routinely a 5 µl aliquot of each eluate (1/24) was finally amplified in a subsequent real-time PCR to detect BVDV infection from the pools, down to 10 genomic copies per reaction and is in the range of sensitivities required by the government. This corresponds approximately to a dilution of a persistently infected animal (PI) sample of at least 1:1000. (Data kindly provided from Dr. Nieper; LUA Leipzig) Fig. 12 Isolation and detection of the Respiratory Syncytial Virus (RSV) from pharyngeal swab Virus RNA was isolated from RSV infected pharyngeal swab sample using the InviMag® Virus DNA/RNA Mini Kit/ KFmL. The samples were analyzed using a RSV specific RT-PCR. 10 µl of eluted RSV - RNA was used per cDNA synthesis and was analyzed on a 2 % agarose gel. lane 1 lane 2/3 lane 4 lane 5 1 2 3 4 5 100 bp Ladder (Gibco) no sample positive control Respiratory Syncytial Virus (RSV) infected patient sample (Data kindly provided from Dr. H. Hannig, Central Institution for Molecular Diagnostic, Clinic Braunschweig gGmbH) 15 Detection of internal controls For the detection of the Internal Control provided in the RTP® kits, a special Control Detection Assay is available for use on established real-time PCR instruments. The assay includes all primers, probes (FAM, TAMRA) and enzyme mix for convenient use. The DNA Control Detection Assay detects internal DNA Extraction Controls and monitors the viral DNA purification. The assay has a detection limit of ≤ 5 DNA copies and the limit of detection depends on sample matrix, processing grade, DNA preparation and DNA content. Sample Mean Ct value Internal DNA Extraction Control Prep 1 27.44 Internal DNA Extraction Control Prep 2 27.92 Internal DNA Extraction Control Prep 3 27.68 Internal DNA Extraction Control Prep 4 27.61 Internal DNA Extraction Control Prep 5 28.24 Internal DNA Extraction Control – Positive Control 26.22 Internal DNA Extraction Control – Negative Control HBV Prep 1 38.34 HBV Prep 2 35.57 HBV Prep 3 34.18 HBV Prep 4 35.78 HBV Prep 5 35.89 HBV – Positive Control 29.89 Fig. 15 DNA Control Detection Assay Five different HBV containing serum samples were extracted using the RTP® DNA/RNA Virus Mini Kit. HBV was detected by a real-time method, using 5 µl of template DNA, with an in-house PCR assay. Extraction controls were measured using the DNA Control Detection Assay, using 5 µl of template DNA, according to the protocol. The experiment was performed on a Corbett Rotor-Gene™ 3000 instrument. All internal DNA extraction control samples show highly reproducible extraction quality, while HBV titer is varying. HBV – Negative Control Ordering information Product Package Size Catalogue No DNA Control Detection Assay 100 preps 1049110200 16 References ® RTP DNA/RNA Virus Mini Kit Detection of neuropathogenic strains of Equid Herpesvirus 1 (EHV-1) associated with abortions in Germany. Fritsche AK, Borchers K. Vet Microbiol. 2011 Jan 10;147(1-2):176-80 A 12-year molecular survey of clinical herpes simplex virus type 2 isolates demonstrates the circulation of clade A and B strains in Germany. Schmidt-Chanasit J, Bialonski A, Heinemann P, Ulrich RG, Günther S, Rabenau HF, Doerr HW. J Clin Virol. 2010 Jul;48(3):208-11 Diagnostic approach for the differentiation of the pandemic influenza A(H1N1)v virus from recent human influenza viruses by real-time PCR. Schulze M, Nitsche A, Schweiger B, Biere B. PLoS One. 2010 Apr 1;5(4):e9966 Shedding and Transmission of Novel Influenza Virus A/H1N1 Infection in Households—Germany, 2009 Thorsten Suess, Udo Buchholz, Susann Dupke, Roland Grunow, Matthias an der Heiden, Alla Heider, Barbara Biere, Brunhilde Schweiger, Walter Haas, Gérard Krause, and on Behalf of the Robert Koch Institute Shedding Investigation Group. Am. J. Epidemiol., Jun 2010; 171: 1157-1164 Distinction of Influenza B virus lineages Yamagata and Victoria by real-time PCR Barbara Biere, Bettina Bauer, and Brunhilde Schweiger. J. Clin. Microbiol., Jan 2010; 10.1128/JCM.02116-09 Genetic Variability of Group A Human Respiratory Syncytial Virus Strains Circulating in Germany from 1998 to 2007 Janine Reiche and Brunhilde Schweiger. J. Clin. Microbiol., Jun 2009; 47: 1800 – 1810 Molecular Analysis of Varicella-Zoster Virus Strains Circulating in Tanzania Demonstrating the Presence of Genotype M1 Jonas Schmidt-Chanasit et al.; JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2008, p. 3530–3533, Vol. 46, No. 10 ® Invisorb Spin Virus RNA Mini Kit Distribution of different hepatitis C virus genotypes in patients with hepatitis C virus infection. Farah Bokharaei Salim, Hossein Keyvani, Afsaneh Amiri, Fatemeh Jahanbakhsh Sefidi, Ramin Shakeri, Farhad Zamani; World J Gastroenterol 2010 April 28; 16(16): 2005-2009 Spread and interaction of Pepino mosaic virus (PepMV) and Pythium aphanidermatum in a closed nutrient solution recirculation system: effects on tomato growth and yield. Schwarz, D., Beuch, U., Bandte, M., Fakhro, A., Büttner, C. and Obermeier, C. (2010), Plant Pathology, 59: 443–452 Factors associated with death or intensive care unit admission due to pandemic 2009 influenza A (H1N1) infection. Tabarsi P, Moradi A, Marjani M, Baghaei P, Hashemian SM, Nadji SA, Fakharian A, Mansouri D, Masjedi M, Velayati A. Ann Thorac Med 2011;6:91-5 ® Invisorb Spin Virus DNA Mini Kit Occult hepatitis B virus infection in Egyptian hemodialysis patients with or without hepatitis C virus infection; Hisham Ismail, Mohamed Soliman, Nahed Ismail; Pathology and Laboratory Medicine International 2010:2 113–120 17 About STRATEC Molecular STRATEC Molecular – part of the STRATEC group since 2009 – is a globally active provider of innovative system solutions for nucleic acid sample collection, stabilization, and both manual and automated purification from any sample type. Since 1992 the company is internationally respected for its outstanding and high performance technology platforms and offers a broad spectrum of superior products for molecular diagnostics, drug discovery and Life Science research. As an EN ISO 13485:2003 + AC 2007 and EN ISO 9001:2008 certified company all STRATEC Molecular products are subject to extensive quality control. In compliance with Directive 98/79/EC (IVDD) many STRATEC Molecular products are CE-certified. STRATEC Molecular guarantees the correct function of all products and highest quality support by first-rate service. About the STRATEC group The STRATEC group consists of the publicly listed parent company STRATEC Biomedical AG and of subsidiaries and second-tier subsidiaries in Germany, the USA, the UK, Switzerland and Romania. The STRATEC Biomedical AG (http://www.stratec.com) designs and manufactures fully automated systems for its partners in the fields of clinical diagnostics and biotechnology. Core technologies ® InviTrap - Selective removal of DNA Non-chaotropic chemistry - shorter protocols through reduced salt concentrations higher yields from complex/precious samples more intact chromosomal DNA - - ® ® InviMag - Magnetic beads MSB - Minimal salt binding - - the fastest way to purify DNA fragments excellent purity without washing - RNAsure - Viral RNA protection - Extraction Tube provides all lysis components, Carrier RNA and standards stabilized at RT safer sample handling due to reduced hands-on steps - ® PSP - DNA sample stabilization - manual and automated DNA and RNA purification ® ® RTP - Ready to prep - highly purified RNA for better RT-PCR results no DNase digestion required stabilization of host/pathogen DNA at RT in stool, saliva or swab samples preservation of bacterial titers at the time of sampling 18 immediate lysis and viral RNA stabilization in biological samples RNA resistant to degradation for up to 6 month at RT
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