PCRopsisTM Reagent RVD For In Vitro Diagnostic Use

REF #: 783025 & 783100

Store at room temperature

INTENDED USE PCRopsis™ Reagent RVD is intended for extraction-free amplification of RNA from RNA viruses collected using a nasopharyngeal or oropharyngeal swab and transported in compatible transport mediums within 7 days from collection and properly stored.

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01 INTRODUCTION PCRopsis™ Reagent RVD is engineered to simultaneously bind a variety of reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) inhibitors found in RNA viral samples, lyse RNA viruses in transport medium and stabilize RNA in a manner that's compatible with RTqPCR. The product consists of a proprietary mixture of salts, stabilizers, buffers and sodium azide to achieve this task, without employing detergents. Reagent RVD allows for extraction-free 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.

02 PRODUCT SIZE

CatalogNumber Volume

783025 25mL

783100 100mL

03 STORAGE & STABILITY

PCRopsis™ Reagent RVD is shipped and stored at room temperature. The recommended storage temperature is: 4°C ~ 25°C

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04 TRANSPORT MEDIUM COMPATIBILITY

RECOMMENDED:

•  PCRopsis™ Universal Viral Transport (UVT) – preferred

•  MedSchenker Smart Transport Medium - preferred

•  BD™ Universal Viral Transport System (UVT)

•  Quest V-C-M Medium

•  Puritan UniTranz-RT Universal Transport Solution

•  Remel MicroTest™ M6™ Transport Medium

•  TransPRO CVM™ Transport Medium

•  Mediums with recipes similar to PCRopsis™ UVT are expected to be compatible

•  Copan ESwab™ Amies Medium

NOT RECOMMENDED:

u  Phosphate buffered saline (PBS)

u  Mediums containing guanidinium thiocyanate

u  Hank's Balanced Salt Solution (HBSS) + 2% Fetal Bovine Serum (FBS)

u  Water with veal infusion broth + BSA + antibiotics

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05 OVERVIEW OF PROTOCOL

Reagent RVD

Viral test sample in Universal Viral

Transport medium

Mix 50 μL Reagent RVD + 50 μL Sample /

well

Heat Reagent RVD + Sample mixture to 95°C for 10 minutes

Use 5 μL of processed sample into your desired RT-qPCR mixture

NOTE: samples can be heated in a

thermal cycler or heating block

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06 WRITTEN PROTOCOL

1.  Thoroughly mix Reagent RVD to ensure homogeneity, but avoid creating bubbles unnecessarily

1.  Reagent RVD 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 (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)

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07 STEP-BY-STEP PROTOCOL WITH FIGURES

Step 1

Gently invert Reagent

RVD to ensure

homogeneity

Step 2

Add Reagent RVD to

reservoir

Step 3

Add 50 μL of Reagent

RVD to wells in a 96-

well PCR plate PCRopsisTM Reagent RVD 6 of 10

Step 4 Step 5

Seal 96-well plate with

a plate sealer to

prevent evaporation

Step 6

Mix 50 μL of viral sample

to wells containing 50 μL

of Reagent RVD

07 STEP-BY-STEP PROTOCOL WITH FIGURES

Heat Reagent RVD +

sample mixture to 95°C

for 10 minutes

Pre-heated thermal cycler or heating block before

applying plate

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Step 7 Step 8

Mix 5 μL of heated Reagent

RVD + sample mixture with

your RT-qPCR mix

Step 9

Add ~15 μL RT-qPCR mix

from your desired vendor

to a new plate

07 STEP-BY-STEP PROTOCOL WITH FIGURES

Detect amplification of

target genes using your

desired qPCR equipment PCRopsisTM Reagent RVD 8 of 10

08 TROUBLESHOOTING & SUGGESTIONS

1.  Reagent RVD is optimized for the amplification of gene targets from RNA viruses in compatible

transport mediums and may not be applicable for other applications or mediums.

2.  For best results, use recently collected viral samples in compatible transport mediums that

have been stored at ~4°C since collection.

3.  Samples are diluted 50% with Reagent RVD. As such, you should expect to observe slightly

higher Ct’s compared to RNA extracted samples.

4.  Ct cut-offs for assays should be increased, often times to 40 cycles.

5.  Take care in maintaining the sterility of your Reagent RVD stock after use.

6.  It’s recommended to immediately use the heated Reagent RVD + sample mixture for

downstream applications.

09 CONTACT

Contact our research team if assistance with Reagent RVD is necessary (info@entopsis.com). We

will try our best to assist with non-intended applications of this product or direct you to alternative

products. Any business related questions should be directed to: Sales@PCRopsis.com.

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7600 NW 69th Ave, Medley, FL 33166

www.PCRopsis.com

info@entopsis.com

sales@PCRopsis.com

Toll-free 1-888-407-5070

PCRopsisTM is a trademark of Entopsis

v.20200827

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