Key Points:

  • PCRopsis™ Reagent RVD-E offers extraction-free amplification of nucleic acids directly from swabs
  • Offers comparable RT-qPCR / PCR results to traditional extraction procedures for many applications 
  • Saves you time, materials, and money
  • Compatible with various synthetic swabs 
  • Cost a fraction of traditional extraction procedures, with fewer steps
  • Compatible with most leading RT-qPCR / PCR kits and master mixes
  • Compatible with automated sample processing equipment
  • Reagent RVD-E is stored and shipped at room temperature

​Intended Use (IN VITRO DIAGNOSTIC USE)
  • PCRopsis™ Reagent RVD-E is intended for extraction-free amplification of RNA and DNA from specimens on swabs, without the need for transport mediums.
  • Reagent RVD-E may replace extraction procedures 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-E
PCRopsis™ Reagent RVD-E is engineered to simultaneously elute material from swabs, bind a variety of RT-qPCR / PCR inhibitors found in clinical samples, lyse specimens and stabilize nucleic acids in a manner that's compatible with RT-qPCR / PCR. The product consists of a proprietary mixture of peptides, salts, stabilizers, buffers and sodium azide to achieve this task. Reagent RVD-E allows for extraction-free amplification of nucleic acids without performing extractions, 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-E for unintended applications.

​Product Deterioration
PCRopsis™ ​ Reagent RVD-E 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.​

​Extraction-Free Swab Protocol
Recommended swabs:
  • Synthetic swabs - rayon, dacron, polyester, nylon (preferred) with aluminum or plastic shafts
Material to be tested:
  • Specimen-containing swab properly transported in a sterile tube
 
  1. Thoroughly mix Reagent RVD-E to ensure homogeneity, but avoid creating bubbles unnecessarily 
    1. Reagent RVD-E has a hazy, white color when homogenized and normal settlement occurs if not regularly mixed
  2. Elute material from swab:
    1. Add 200 μL of Reagent RVD-E to transport tube with swab
    2. Make sure the swab is at least partially submerged into Reagent RVD-E
    3. Vortex for ~40 seconds
  3. Specimen lysis & nucleic acid stabilization:
    1. Transfer ~100 μL of eluted sample into a thin-walled PCR tube / plate and then cap tube or apply plate sealer to plate to prevent evaporation
    2. Heat at 95°C
      1. Mammalian:  5 minutes
      2. Viruses:          10 minutes
      3. Bacteria:        15 minutes
      4. NOTE: heating for a longer period of time does not negatively affect results
    3. Let cool at room temperature for ~10 seconds before continuing
  4. Pipette up & down to ensure complete mixing 
  5. Use 5 - 10 μL of lysed / stabilized sample in your desired RT-qPCR procedure
    1. ​Lysed sample can represent 25%~50% of your final RT-qPCR mixture (i.e., 5 ~ 10 μL sample into a total volume of 20 μL)

Limitations of the Procedure
  • Performance characteristics of Reagent RVD-E were validated using SARS-CoV-2, S. aureus and P. aeurginos with flocked nylon swabs by RT-qPCR. The use of alternative microorganisms, swabs, gene targets and / or detection methods may affect the performance of the product.
  •  Repeated freezing and thawing of test specimens or over drying swabs 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-E 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-E enables consistent amplification of RNA and DNA gene targets derived from viruses (e.g., coronaviruses) or bacteria (e.g., S. aureus) adhered to synthetic swabs without the need for nucleic acid extraction procedures. Each lots is carefully tested for sterility and functionality to ensure quality and consistent results.

Certificate of Analysis:
LOT3     LOT4

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 mediums 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-E

Reagent RVD-E + RT-qPCR correctly identifies 100% of tested COVID-19 samples and amplifies human control gene RNase P

Reagent RVD-E mediates extraction-free amplification of N1, N2 and human RNase P gene sites from clinical samples. SARS-CoV-2 genes N1 and N2 were amplified in all positive samples and no amplification was observed in negative samples. 

Reagent RVD-E + RT-qPCR has a limit of detection of <53 viral genome copies / RT-qPCR reaction for SARS-CoV-2

Reagent RVD-E facilitates viral RNA extraction-free amplification of N1 and N2 sites on heat inactivated SARS-CoV-2 dried onto nylon flocked swabs. Both gene sites were amplified in samples containing as little as 53 viral genome copies / RT-qPCR reaction. Samples were tested in duplicates.
Reagent RVD-E + PCR has a limit of detection of <100 cfu / mL for gram positive and negative bacteria 
Reagent RVD-E facilitates extraction-free detection of gram positive and negative bacteria dried onto nylon flocked swabs, with a limit of detection <100 cfu / mL. Samples were tested in duplicates.

Satisfaction Guarantee - Warranty Summary

Entopsis guarantees the performance of Reagent RVD-E to facilitate extraction-free amplification of tested coronaviruses and bacteria using RT-qPCR from samples dried onto flocked nylon swabs and properly stored. Within 7 days of purchase, contact Entopsis (info@entopsis.com) should Reagent RVD-E 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-E 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-E for its particular use. However, the Entopsis scientific team can assist in the optimization of Reagent RVD-E 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-E are offered. 

​THIS WARRANTY ONLY APPLIES IF PRODUCTS ARE PURCHASED FROM AN APPROVED VENDOR (E.G., ENTOPSIS).

Complete Terms and Conditions
PCRopsis: part of the Entopsis family
info@entopsis.com