Key Points:
|
|
Intended Use (IN VITRO DIAGNOSTIC USE)
PCRopsis™ Reagent RVD-RT is intended for extraction-free amplification of RNA or DNA from properly collected and transported specimens in compatible transport mediums at room temperature.
- Reagent RVD-RT may replace nucleic acid extraction procedures for many applications (e.g., life science research, veterinary pathogen research, etc.), but it must be verified by the end user.
PCRopsis™ Reagent RVD-RT is an alternative for:
- Qiagen: QIAamp Viral RNA mini Kit
- Qiagen: QIAmp DSP Viral RNA Mini Kit
- Qiagen EZ1 Advanced XL: EZ1 DSP Virus Kit
- Qiagen EZ1 Advanced XL: EZ1 Virus Mini Kit v2.0
- QIAGEN QIAcube: QIAmp DSP Viral RNA Mini Kit
- QIAGEN QIAcube: QIAamp Viral RNA Mini Kit
- Roche MagNA Pure 24: MagNA Pure 24 Total NA Isolation Kit
- Roche MagNA Pure 96: DNA and Viral NA Small Volume Kit
- Roche MagNA Pure LC: Total Nucleic Acid Kit
- Roche MagNA Pure Compact: Nucleic Acid Isolation Kit I
- bioMérieux NucliSENS® easyMAG®
- bioMérieux EMAG®
- Thermo Fisher / Applied Biosystems: MagMAX mirVana Total RNA Isolation Kit
- Promega: Direct PCR: XpressAmp™ Direct Amplification Reagents
The Science Behind Reagent RVD-RT
PCRopsis™ Reagent RVD-RT, with PCRopsis™ Activator, is engineered to simultaneously bind a variety of RT-qPCR / PCR inhibitors found in clinical specimens, lyse viruses / mammalian cells, and stabilize RNA and DNA in a manner that's compatible with RT-qPCR / PCR. The product consist of a proprietary mixture of peptides, salts, stabilizers, buffers, RVD Enhancer, and sodium azide to achieve these tasks. Reagent RVD-RT allows for extraction-free amplification of viral / mammalian RNA / DNA without performing nucleic acid isolation, centrifugations or other sample manipulations which may introduce errors, contaminants and/or skew the representation of RNA fragments.
User should verify the applicability of Reagent RVD-RT for unintended applications.
User should verify the applicability of Reagent RVD-RT for unintended applications.
Reagent RVD-RT vs Old Fashion RNA Extraction
Traditional RNA Extraction Results in Significantly Different Levels of miRNA ... this bias can affect results.
Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal
Rikki A. M. Brown, Michael R. Epis, Jessica L. Horsham, Tasnuva D. Kabir, Kirsty L. Richardson & Peter J. Leedman BMC Biotechnology volume 18, Article number: 16 (2018) - Link to manuscript
A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma
Ryan K.Y. Wong, Meabh MacMahon, Jayne V. Woodside & David A. Simpson BMC Genomics volume 20, Article number: 446 (2019) - Link to manuscript
RNA Preservation Agents and Nucleic Acid Extraction Method Bias Perceived Bacterial Community Composition
Ana McCarthy, Edna Chiang, Marian L. Schmidt, Vincent J. Denef, PLOS ONE (2015) - Link to manuscript
Rikki A. M. Brown, Michael R. Epis, Jessica L. Horsham, Tasnuva D. Kabir, Kirsty L. Richardson & Peter J. Leedman BMC Biotechnology volume 18, Article number: 16 (2018) - Link to manuscript
A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma
Ryan K.Y. Wong, Meabh MacMahon, Jayne V. Woodside & David A. Simpson BMC Genomics volume 20, Article number: 446 (2019) - Link to manuscript
RNA Preservation Agents and Nucleic Acid Extraction Method Bias Perceived Bacterial Community Composition
Ana McCarthy, Edna Chiang, Marian L. Schmidt, Vincent J. Denef, PLOS ONE (2015) - Link to manuscript
Reagent RVD-RT does not use "capture & release" RNA / DNA technology (e.g., columns, beads, etc.), therefore it does not exhibit this bias.
Studies with PCRopsis™ Reagent RVD-RT (containing PCRopsis™ Activator)
PCRopsis™ Reagent RVD-RT significantly outperforms Promega XpressAmp at room temperature Direct PCR: SARS-CoV-2 in Transport Mediums
Various viral transport mediums were spiked with SARS-CoV-2 and processed with Reagent RVD-RT and Promega XpressAmp direct PCR reagent according to the manufacturer's protocols. RT-qPCR was performed on processed samples using primers and probes for four SARS-CoV-2 gene targets (N1, N2, E, N(Japan)). Reagent RVD-RT consistently resulted in lower Ct values for most tested viral transport mediums and gene targets compared to Promega XpressAmp direct PCR reagent (highlighted in red). Moreover, Promega's direct PCR reagent often resulted in no Ct values for several transport mediums and gene targets, thus producing high levels of false negatives.
RNA was extracted using Qiagen QIAamp Viral RNA Kit. RT-qPCR was performed using IDT qPCR probe assay and Promega GoTaq® Probe 1-Step RT-qPCR System. Each condition was tested in duplicates and averaged.
RNA was extracted using Qiagen QIAamp Viral RNA Kit. RT-qPCR was performed using IDT qPCR probe assay and Promega GoTaq® Probe 1-Step RT-qPCR System. Each condition was tested in duplicates and averaged.
PCRopsis™ Reagent RVD-RT significantly outperforms Promega XpressAmp at room temperature Direct PCR: SARS-CoV-2 in Saliva
RNA was extracted using Qiagen QIAamp Viral RNA Kit. RT-qPCR was performed using IDT qPCR probe assay and Promega GoTaq® Probe 1-Step RT-qPCR System. Each condition was tested in duplicates and averaged.
|
Human saliva was spiked with SARS-CoV-2 and processed with Reagent RVD-RT and Promega XpressAmp direct PCR reagent according to the manufacturer's protocols. RT-qPCR was performed on processed samples using primers and probes for three SARS-CoV-2 gene targets (N1, N2, E) and one human control gene target (RNaseP). Reagent RVD-RT consistently resulted in lower Ct values for all tested gene targets compared to Promega XpressAmp direct PCR reagent (highlighted in red). Promega's direct PCR reagent resulted in Ct values that were 4 ~ 12 Ct greater than Reagent RVD-RT. Reagent RVD-RT resulted Ct values comparable or lower than extracted RNA.
|
PCRopsis™ Reagent RVD-RT + RT-qPCR detects 3.9 viral copies / µL from transport medium containing SARS-CoV-2
|
Transport medium was spiked with inactivated SARS-CoV-2 (ATCC) and processed with Reagent RVD-RT (with Activator). Five viral gene sites were amplified through RT-qPCR, in duplicates, and averages shown. Reagent RVD-RT mediated amplification of tested gene regions with Ct values between 34 ~40 at 3.9 viral copies / µL.
RT-qPCR was performed using IDT qPCR probe assay and Promega GoTaq® Probe 1-Step RT-qPCR System. |
PCRopsis™ Reagent RVD-RT amplifies <50 CFU / mL of gram-positive / negative bacteria in transport medium
|
Transport medium was spiked with bacteria and processed with Reagent RVD-RT for 10 minutes at room temperature. The reagent mediated extraction-free PCR, even when samples were diluted to under 50 cfu / mL.
qPCR was performed using IDT qPCR primers / probe and PCRopsis™ 5x PCR Master Mix. Each condition was tested in duplicates and averaged. |
Satisfaction Guarantee - Warranty Summary
Entopsis guarantees the performance of Reagent RVD-RT to facilitate the direct amplification of select viruses and mammalian cells using PCR / RT-qPCR from saliva, urine and swab samples in compatible mediums when the sample is properly stored. Within 7 days of purchase, contact Entopsis (info@entopsis.com) should Reagent RVD-RT fail to perform as described above or in the CoA. In such a scenario, Entopsis will re-test the Lot in question using remaining reagent at our facility at our expense (purchaser does not need to ship product to Entopsis). If the re-tested Lot fails to meet our performance criteria, Entopsis will replace the faulty reagent free of charge. If the Lot meets our performance criteria as stated on the Certificate of Analysis for the Lot in question, then no additional action will be taken.
Please keep in mind that purchasers may use Reagent RVD-RT for numerous applications, sample types, gene targets, conditions, etc. that are untested and thereby may not work as desired. The purchaser must determine the suitability of Reagent RVD-RT for their particular use. However, the Entopsis scientific team can assist in the optimization of Reagent RVD-RT for your desired application. Refunds will not be provided due to a lack of product compatibility with purchaser's desired application. This is why small test volumes of Reagent RVD-RT are offered.
THIS WARRANTY ONLY APPLIES IF PRODUCTS ARE PURCHASED FROM AN APPROVED VENDOR (E.G., ENTOPSIS).
Complete Terms and Conditions
Please keep in mind that purchasers may use Reagent RVD-RT for numerous applications, sample types, gene targets, conditions, etc. that are untested and thereby may not work as desired. The purchaser must determine the suitability of Reagent RVD-RT for their particular use. However, the Entopsis scientific team can assist in the optimization of Reagent RVD-RT for your desired application. Refunds will not be provided due to a lack of product compatibility with purchaser's desired application. This is why small test volumes of Reagent RVD-RT are offered.
THIS WARRANTY ONLY APPLIES IF PRODUCTS ARE PURCHASED FROM AN APPROVED VENDOR (E.G., ENTOPSIS).
Complete Terms and Conditions