Key Points:

Compatible with WHO microbial transport medium (water + veal infusion broth + BSA + antibiotics)​
  • Reagent RVD-WHO is an alternative to RNA extraction for the amplification of viral RNA 
  • Offers comparable RT-qPCR results to RNA extraction procedures for many applications in ~10 MINUTES
  • Compatible with nasopharyngeal and oropharyngeal swabs
  • Cost a fraction of traditional RNA extraction procedures, with fewer steps
  • Compatible with leading microbial transport mediums
  • Compatible with most leading RT-qPCR kits and master mixes
  • Compatible with automated sample processing equipment
  • Reagent RVD-WHO is stored and shipped at room temperature

​Intended Application (IN VITRO DIAGNOSTIC USE)
  • PCRopsis™  Reagent RVD-WHO is intended for extraction-free amplification of RNA from RNA viruses collected using a nasopharyngeal or oropharyngeal swab and properly transported in WHO microbial transport medium (water + veal infusion broth + BSA + antibiotics).
  • Reagent RVD-WHO may replace viral RNA extraction for many applications (e.g., life science research, veterinary pathogen research, etc.), but it must be verified by the end user.

​The Science Behind Reagent RVD
PCRopsis™  Reagent RVD-WHO is engineered to simultaneously bind a variety of RT-qPCR inhibitors found in RNA viral samples (e.g., coronaviruses) transported in WHO microbial transport medium, lyse RNA viruses and stabilize RNA in a manner that's compatible with RT-qPCR. The product consist of a proprietary mixture of salts, stabilizers and buffers to achieve this task, without employing detergents. Reagent RVD-WHO allows for direct amplification of viral RNA without performing RNA isolation, centrifugations or other sample manipulations which may introduce errors, contaminants and/or skew the representation of RNA fragments.
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User should verify the applicability of Reagent RVD-WHO for unintended applications.

​Product Deterioration
PCRopsis™ ​ Reagent RVD-WHO should not be used if (1) there is evidence of damage or contamination to the product, (2) there is evidence of leakage, (3) the color of the reagent has changed from clear-white hazy, (4) the expiration date has passed, or (5) there are other signs of deterioration.​

Reagent RVD-WHO vs 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
Reagent RVD-WHO does not use "capture & release" RNA / DNA technology (e.g., columns, beads, etc.), therefore it does not exhibit this bias.

​Extraction-Free Protocol
Recommended transport medium:
  • WHO microbial transport medium (water + veal infusion broth + BSA + antibiotics)

Not Recommended Transport Mediums:
  • Transport mediums other than WHO transport medium (water + veal infusion broth + BSA + antibiotics)
  • Phosphate buffered saline (PBS)
  • Mediums containing guanidinium thiocyanate
  • Hank's Balanced Salt Solution (HBSS) + 2% Fetal Bovine Serum (FBS)
 
  1. Thoroughly mix Reagent RVD-WHO to ensure homogeneity, but avoid creating bubbles unnecessarily 
    1. Reagent RVD-WHO has a hazy, white color when homogenized and normal settlement occurs if not regularly mixed
  2. Mix 1 volume of sample (50 μL) with 1 volume of Reagent RVD-WHO (50 μL) in a thin walled tube (0.2 ~ 0.6 mL) or 96-well PCR plate
  3. Pipette up & down to ensure complete mixing and then cap tube or apply plate sealer to plate to prevent evaporation
  4. Heat diluted sample for 10 minutes at 95°C and let cool at room temperature for ~10 seconds before continuing 
    1. Alternatively, you can heat the diluted sample for 15 minutes at 80°C
    2. Make sure the heating device has reached the desired temperature before applying sample
    3. Sample heating can be performed using a controlled heating block or thermal cycler
  5. Mix heated sample and use 1 - 5 μL of lysed / stabilized sample in your desired RT-qPCR procedure
    1. ​25% of lysed sample can be added to your RT-qPCR mixture (i.e., 5 μL sample into a total volume of 20 μL)

Limitations of the Procedure
  • Performance characteristics of Reagent RVD-WHO was validated using SARS-CoV-2 nasopharyngeal swab samples in WHO microbial transport medium, where the N1 and N2 gene sites were amplified by RT-qPCR using Promega GoTaq Probe 1-Step RT-qPCR System. The use of alternative viruses, transport mediums, gene targets and / or detection methods may affect the performance of the product.
  • Repeated freezing and thawing of test specimens may reduce the detection of desired gene targets.
  • Follow recommended guidelines for specimen collection as this may affect the ability to detect desired organisms.

PCRopsis Reagent RVD-WHO is an alternative for:
  1. Qiagen: QIAamp Viral RNA mini Kit
  2. Qiagen: QIAmp DSP Viral RNA Mini Kit
  3. Qiagen EZ1 Advanced XL: EZ1 DSP Virus Kit
  4. Qiagen EZ1 Advanced XL: EZ1 Virus Mini Kit v2.0
  5. QIAGEN QIAcube: QIAmp DSP Viral RNA Mini Kit 
  6. QIAGEN QIAcube: QIAamp Viral RNA Mini Kit
  7. Roche MagNA Pure 24: MagNA Pure 24 Total NA Isolation Kit
  8. Roche MagNA Pure 96: DNA and Viral NA Small Volume Kit 
  9. Roche MagNA Pure LC: Total Nucleic Acid Kit 
  10. Roche MagNA Pure Compact: Nucleic Acid Isolation Kit I 
  11. bioMérieux NucliSENS® easyMAG®
  12. bioMérieux EMAG®
  13. Thermo Fisher / Applied Biosystems: MagMAX mirVana Total RNA Isolation Kit

​Performance & CoA
PCRopsis™  Reagent RVD-WHO enables consistent amplification of RNA gene targets derived from RNA viruses (e.g., coronaviruses) when transported in WHO microbial transport medium without the need for RNA extraction procedures. Each lots is carefully tested for sterility and amplification of RNA in transport medium to ensure quality and consistent results.

Results obtained will largely depend on the use of compatible sample transport medium and gene target being amplified. 

Certificate of Analysis:
LOT2

Storage

This product is ready for use and no further preparation is necessary. The product should be transported and stored in its original container at 4–25°C until used. Do not overheat. Do not incubate or freeze prior to use. Improper storage will result in a loss of efficacy. Do not use after expiration date, which is clearly indicated on the bottle.
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Warnings & Precautions

FOR IN VITRO DIAGNOSTIC USE 
  • Observe approved biohazard precautions and aseptic techniques to prevent contamination of the product. To be used only by adequately trained and qualified personnel.
  • Pathogenic microorganisms, including hepatitis viruses and Human Immunodeficiency Virus, may be present in clinical specimens. "Standard Precautions" and institutional guidelines should be followed in handling all potentially bio-hazardous materials.
  • Sterilize all biohazard waste including specimens, containers and medium after their use.
  • Directions should be read and followed carefully.
  • Do not re-pack.
  • The use of this product in association with a rapid diagnostic kit, diagnostic instrumentation or used in a manner not intended should be validated by the user.
  • Do not ingest the reagent.
  • Avoid skin contact with reagent since it contains sodium azide to prevent microbial growth.
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Studies with PCRopsis Reagent RVD-WHO

Reagent RVD-WHO identifies 100% of positive and negative Coronavirus  samples

Viral samples processed with PCRopsis™ Reagent RVD-WHO and traditional RNA extraction followed by RT-qPCR identify 100% of negative and positive samples.

NOTE: The Ct threshold for RT-qPCR is set to 40 cycles with Reagent RVD-WHO.

Satisfaction Guarantee - Warranty Summary

Entopsis guarantees the performance of Reagent RVD-WHO to facilitate the direct amplification of coronaviruses using RT-qPCR from nasopharyngeal samples collected in WHO microbial transport medium (water + veal infusion broth + BSA + antibiotics) and the sample is stored properly. Within 7 days of purchase, contact Entopsis (info@entopsis.com) should Reagent RVD-WHO 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 or refund the purchase price.

Please keep in mind that purchasers may use Reagent RVD-WHO 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 the Reagent RVD-WHO for its particular use. However, the Entopsis scientific team can assist in the optimization of Reagent RVD-WHO 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-WHO are offered. 

THIS WARRANTY ONLY APPLIES IF PRODUCTS ARE PURCHASED FROM AN APPROVED VENDOR (E.G., ENTOPSIS OR RSS MEDICAL DISTRIBUTORS).
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Complete Terms and Conditions
PCRopsis: part of the Entopsis family
info@entopsis.com