myPOLS 6901 Volcano 3G Direct COVID-19 Kit Instruction Manual

myPOLS 6901 Volcano 3G Direct COVID-19 Kit

Intended Use

Volcano3G® Direct COVID-19 Kit contains the components for a multiplex real- time reverse transcription (RT)-PCR for the qualitive detection of SARS-CoV-2 RNA in human gargle solution. On the one hand the RNA from the gargle solution can be extracted with a separate extraction kit and applied in RT-PCR. On the other hand, the kit is intended to apply the gargle solution direct in RT-PCR, without RNA extraction. The kit is designated for the application on a QuantStudioTM 5 (Thermo Fisher), LightCycler® 480 II (Roche) and CFX96 Touch (Bio-Rad) instrument or an appropriate real-time PCR cycler.
The RT-PCR can be applied with gargle solution of persons that do not show symptoms or do show symptoms of COVID-19. Results are for the detection of SARS-CoV-2 RNA. This supports the diagnosis of infections with SARS-CoV-2. Generally, SARS-CoV-2 RNA can be detected in respiratory specimens during the acute phase of infection. Positive results are indicative for the presence of SARS-CoV-2 RNA, but do not exclude a bacterial infection or a co-infection with other viruses. The positive detection of SARS-CoV-2 RNA may not be the cause of disease. It may be required that laboratories report positive results to the appropriate Competent Health Authorities.
Negative results do not exclude an infection with SARS-CoV-2 and should not be used as sole basis for further treatment steps of a patient.
The product is not designed for self-testing but is intended for the professional application by qualified persons trained in techniques for sample processing, RT-PCR and in-vitro diagnostic procedures.

Product Description

The Kit is intended for the detection of SARS-CoV-2 RNA in a multiplex real- time RT-PCR from human gargle solution. Gargle solution (see Chapter 6.) is applied as specimen that can be used extracted or directly in PCR.
The kit contains the following components:

• Ready-to-use master mix (Volcano3G® RT-PCR Probe 2x Master Mix: hotstart-formulated DNA polymerase, dNTPs, buffer components, PCR-additives and a blue dye as pipetting control)
• Duplex assay containing primer and probes for binding to N-gene of SARS-CoV-2 RNA and binding to human gene for RNaseP as internal control
• Positive control containing plasmid DNA for target region of SARS-CoV-2 and target region of RnaseP
•  Direct Booster Solution as PCR enhancer for applying the gargle solution directly without RNA extraction

Contents

Aliquot size for 100 reactions, #6901
Table 1 : Volcano3G® Direct COVID-19 Kit, 100 reactions.

Additional required equipment and materials (not provided):
RT-qPCR-components:

• PCR-grade, RNase-free water

General laboratory equipment

• Disposable powder-free gloves (latex or nitrile)
• Biosafety cabinet when working with infectious/bio-hazardous/unknown samples
• Laboratory freezer (-25 to -15 °C), laboratory fridge (4 – 12 C)
• Adjustable pipettes with sterile, RNase-free filter tips
• RNase-free Microcentrifuge tubes
• RNA extraction kit (optional)
• Ice or cooling rack
• Vortex mixer
• Desktop centrifuge (for tubes and plates)
• PCR tubes or -plates with appropriate lid or seal
• Real-time PCR cycler with analysis software and computer

Assay limitations

• This product is intended to be used for diagnostic assays performed by qualified and trained laboratory personnel. The product is not intended to be used for self-testing.
• Follow good laboratory practices as well as safety notes and guidelines described in this instruction for use. Read the instructions for use carefully before using the product.
• The quality of the gargle solution as well as the extracted RNA from the gargle solution is important for the functionality of the product. The performance of the kit was established and evaluated with human gargle solution applied directly or with extracted RNA. The application of other specimens was not tested and should not be analyzed with this kit.
• Specimens must be collected, transported and stored under appropriate conditions. Improper handling of the specimens may prevent the detection of SARS-CoV-2 RNA.
• Negative results do not exclude an infection with SARS-CoV-2 and should not be used as sole basis for further treatment steps of a patient.
• Before collecting the samples, the test person must not have eaten, drunk or smoked for at least 30 min and must not have brushed his teeth or used a mouth rinsing solution.

Storage and Handling

Volcano3G® Direct COVID-19 Kit is shipped on cool packs (<10 °C). After receipt Volcano3G® RT-PCR Probe 2x master mix, 10x S-CoV2 Duplex Assay and S-CoV2 positive control must be stored at -25 °C to -15 °C. Direct Booster solution must be stored at 4 to 12 °C. Only when properly stored, the product can be used until the stated expiration date. Aliquots of Volcano3G® RT-PCR Probe 2x master mix, 10x S-CoV2 Duplex Assay and S-CoV2 positive control for day-to-day use can be stored at 0 – 10 °C. Storage at higher temperatures should be avoided. Freeze-thaw-cycles may harm the products performance. Protect the components from light. To avoid contaminations, it is recommended to store the mix in aliquots and work in a sterile environment.

General Safety Notes

• Read the safety instructions carefully before using the product.
• Do not eat, drink, smoke, or apply cosmetic products in the work areas.
• Treat all specimens and controls as if infectious/biohazardous unless they are known to be not.
• Wear appropriate personal protective equipment when handling specimens.
• Use biosafety cabinet when handling infectious/biohazardous/unknown specimens.
• Follow local regulatory guidelines for waste disposal.
• Use sterile, DNA/DNase/RNase-clean consumables to avoid contaminations.
• Clean all surfaces and equipment in direct environment before starting the experiment to lower the risk of nucleic acid-, DNase/RNase- and protease contaminations.
• Read the instructions for use of all employed Kits and components carefully before the experiments.
• Do not use this product after its expiration date.
• Store the product as specified in Chapter 4.
• Dispose the product if the components leaked due to damage of the primary packaging.
• Modifications of the kit components or assay protocols are possible in principle but are not verified or validated.
• Positive results are indicative for the presence of SARS-CoV-2 RNA.

Specimen Collection, Transport and Storage

• Gargle solution: 5 ml PCR-grade water or 0.9% NaCl solution.
• Collection: Gargling 20 – 30 sec with the gargle solution by the subject.
• The subject spits the gargle solution into PCR-grade tube that is eventually tightly closed.
• Gargling solution can be shipped at room temperature.
• The gargle solution can be stored at 0 to 12 °C or -25 to -15 °C.
• Note: Treat all specimens as if infectious/biohazardous unless they are known to be not.

Direct Application (non-extracted, gargle solution)

• Heat the solution 3 min at 95 °C immediately before use.
• After cool down, 6 µl per 30 µl reaction are directly applied in PCR. The detailed procedure is described in Chapter 7.
• The heat-inactivated gargle solution can be stored 3 h maximum at 0 to 12 °C before use. But the application immediately after heat inactivation is recommended.

RNA Extraction

• RNA is extracted with any standard extraction procedure from not-heat-inactivated gargle solution. The manufacturer´s instructions must be considered.
• The quality of the RNA is important for the functionality of the product. Therefore, it is recommended to validate the RNA extraction procedure before testing.
• 5 µl per 20 µl reaction are applied in PCR. The detailed procedure is described in Chapter 7.
• The extracted RNA should be used immediately after extraction. The quality of the RNA is important for the functionality. Therefore, it is recommended to evaluate the storage conditions of extracted RNA.

Preparation

• All components are stored on ice until their use.
• The PCR-Mix and the PCR-plate can be prepared at room temperature. The DNA polymerases are hotstart-formulated with a specific aptamer. This aptamer prevents unspecific side reactions at room temperature. From 57 °C the aptamer denatures and releases the enzymes.
• The PCR-plate is placed in the cycler immediately after preparation and the protocol is started.
• Work in an RNase-free environment.
• To avoid contaminations, it is recommended to work with a PCR-cabinet or in a similar environment.
• Use filter tips and unique tips for each sample/ control.
• Protect the 10x S-CoV2 Duplex Assay from light.
• Include minimum one positive and one negative (PCR-grade water instead of template) control for each plate to confirm the validity of the assay and to exclude contaminations of the reagents.
• It is recommended to seal the negative controls before the positive control is pipetted to avoid contaminations.
• Familiarize yourself with the general and specific safety guidelines that may be required for the used specimens before starting the experiment.
  1. Thaw the frozen components of the Volcano3G® Direct COVID-19 Kit on ice.
  2. Mix and spin down all components.
  3. Prepare the specimens as described in Chapter 6.
  4. Preparation PCR Master mix: The number of total reactions including positive and negative control is calculated. It is recommended to prepare one additional reaction to compensate pipetting loss. Mix and spin down the components.
     Gargle solution: Mix the components as shown in Table 2.

Table 2: Preparation of the PCR Master Mix for RT-qPCR using gargle solutions.

Extracted RNA: Mix the components as shown in Table 3.
Table 3: Preparation of the PCR Master Mix for RT-qPCR using extracted RNA.

Preparation of the PCR plate

Gargle solution: Pipette the PCR Master Mix into each well of the PCR-plate. Then, add the sample, the positive and negative control (PCR-grade water) according to Table 4.
Table 4: PCR-Mix for using gargle solution. Extracted RNA: Pipette the PCR Master Mix into each well of the PCR-plate. Then, add the sample, the positive and negative control (PCR-grade water) regarding Table 5.
Table 5: PCR-Mix for using extracted RNA.

• Seal the plate with an appropriate seal or lid. Mix and spin down the reactions so that no liquid is seen at the top of the seal or lid.
• The plate is placed in the cycler immediately after preparation and the PCR protocol with the setup described in Chapter 8 is started.

Setup of the real-time PCR Instrument

• * The performance of the kit was established with Bio-Rad CFX96 Dx and LightCycler® 480 II.

  • The PCR-cycler must be technically adjusted in such a way that fluorescence signals can be recorded during PCR in the FAM as well as in the Cy5 channel.
  • Program the PCR protocol given in Table 6 in the software of the real-time PCR instrument (procedure for programming according to the manufacturer’s instructions of the PCR instrument).

Table 6: PCR protocol for using the Volcano3G® Direct COVID-19 Kits. ****

• Set the detections channels for FAM (N-gene of SARS-CoV-2 RNA) and Cy5 (internal control RNaseP) on your cycler.
• The Volcano3G® RT-PCR Probe 2x Master Mix included in the kit does not contain fluorescent reference dye such as ROX. Therefore, make sure that the setting for a reference dye is turned off.

Data Analysis

Quality Control
For data analysis the analysis software of the used real-time PCR instrument according to the manufacturer’s instructions. In some cases, automatic baseline correction by the analysis software might lead to inconsistent baseline behavior and baseline drops below 0 RFU. In this case, please adjust the baseline correction to an appropriate area to obtain consistent results according to the manufacturer’s instructions of the PCR instrument.
Before analyzing the samples, be sure to check the positive and negative control reactions to ensure the results are reliable. For this, the standards given in Table 7 must be fulfilled. All positive controls should demonstrate a distinct amplification during RT-qPCR. The negative controls should not demonstrate any amplification. If the standards give in Table 7 are not fulfilled, the results are inconclusive and the PCR should be repeated. In this case, the results of the samples are not valid. If an amplification is observed in the negative control, check the reagent stocks for contaminations. Furthermore, attention must be paid to critical steps in the workflow where cross-contamination with the sample or the positive control can occur.
Table 7: Standards for a valid RT-qPCR run using Volcano3G® Direct COVID-19 Kit. Positive = distinct amplification detectable, Negative = no amplification detectable.

Volcano3G® Direct COVID-19 Kit – Instructions for use, Version 2, 09/2021

Interpretation of the results
Interpretation of the results is performed as indicated in Table 8. Each sample must demonstrate a distinct amplification in the Cy5-channel (internal control). If not, the results from the FAM-channel for SARS-CoV-2 must be considered invalid. The PCR should be repeated and the quality of the specimen should be checked. If no amplification is detectable after the PCR has been repeated with the same sample, the quality of the specimen is insufficient and a new sample should be collected.
Table 8: Interpretation of the results of the specimen. Positive = distinct amplification detectable, Negative = no amplification detectable.

Analytical Performance

The analytical performance of the Volcano3G® Direct COVID-19 Kits was analyzed by assessing the analytical sensitivity as well as specificity and diagnostic sensitivity as well as specificity as described in the following. The in the following described performance was performed by applying RNA extracts in PCR as well as by applying heat-inactivated gargle solution directly in PCR without RNA extraction.
Analytical Sensitivity (LoD)
For the determination of the limit of detection (LoD) a dilution series of ivRNA (EURM-019, European Commission Joint Research Centre) in buffered aqueous solution (Qiagen AVE-Puffer mit 0.2 µg/µl Poly-A Carrier RNA) was added to reactions containing the Volcano3G® Direct COVID-19 Kit. The samples were analyzed at the LightCycler® 480 II (Roche). The LoD determined from these was subsequently verified with n=40 samples with at least 95 % reliability (Table 9).
Table 9: Results for assessing the analytical sensitivity for the SARS- CoV-2-specific assay of the Volcano3G® Direct COVID-19 Kit.

Analytical Specificity
The analytical specificity of the Volcano3G® Direct COVID-19 Kit is ensured by using primer and probes specified by the CDC. The CDC evaluated in its publication (Emerging Infectious Diseases, Vol.26, No. 8, 2020) the sequences of primer and probes against diverse relevant genome sequences available from the Global Initiative on Sharing All Influenza Data (GISAID). Furthermore, it was shown that no cross reactivity with other respiratory Pathogens occurs.

PCR Cycler Compatibility
In general, a PCR cycler with measuring channels for FAM probes (465-510 nm) and Cy5 probes (618-660 nm) is required. The Volcano3G® Direct COVID-19 Kit was developed and validated by using a LightCycler® 480 II (Roche) und CFX96 Dx (Bio-Rad). The compatibility by using a QuantStudioTM 5 (Thermo Fisher) was shown.

Diagnostic Sensitivity and Specificity
For the determination of the diagnostic sensitivity (Table 10) and specificity (Table 11) 180 samples were analyzed. The samples were clearly defined as positive or negative by using CE-marked reference methods (extraction using Qiagen QIAcube HT oder QIAsymphony and RT-PCR using r-Biopharm RIDA®GENE SARS- CoV-2). According to the manufacturer’s manual of the reference kit the cutoff was set to Ct35.
Using these specifications 60 positive and 120 negative samples were analyzed by applying RNA extracts as well as by applying the samples directly without RNA extraction in combination with the Volcano3G® Direct COVID-19 Kit using a CFX96 Dx (Bio-Rad) or LightCycler® 480 II (Roche).
Table 10: Results for assessing the diagnostic sensitivity by applying gargle solutions directly (without extraction, top) as well as by applying RNA extracts (bottom) in RT-qPCR.

Assessing of the sensitivity using the gargle solutions (Table 10, top) : For samples with medium to high RNA load (reference Ct value < 32), the Volcano3G® Direct COVID-19 Kit shows a sensitivity of 95 %. For samples with low viral RNA load (reference Ct value >32), the diagnostic sensitivity drops below 90%. The simple approach of using gargle solution directly without extraction and the short PCR protocol enables particularly the routine monitoring of larger cohorts to identify highly infectious individuals as quickly as possible and with high specificity. If even the lowest possible viral RNA loads should be reliably detected, extracted samples should be used.
Assessing of the sensitivity using RNA extracts (Table 10, bottom): For clearly positive extracted samples, the kit shows 98% agreement with the reference method. The results can vary depending on the used specimen and extraction method. When inconclusive results are identified, follow-up of possible infection should be performed, taking into account any contacts and symptoms.

For the determination of the diagnostic specificity 120 negative samples defined by the reference method were analyzed (Table 11). The Volcano3G® Direct COVID-19 Kit shows no false-positive results. Thus, the specificity is 100 %.
Table 11: Results for assessing the diagnostic specificity by applying gargle solutions directly (without extraction, left) as well as by applying RNA extracts (right) in RT-qPCR.

Licences/Patents/Disclaimer

This product may not be resold, modified or used for production and commercial purposes of any kind without an agreement with myPOLS Biotec GmbH. For information on obtaining additional rights, please contact: info@mypols.de.
The manufacturer takes no responsibility for possible damages or wrong results originating from the erroneous use of this product.

Key to symbols

References

• Volcano3G® DNA polymerase is based on: Structure and Function of an RNA-Reading Thermostable DNA Polymerase. Angew. Chem. Int. Ed., 2013; 52: 11935–11939. Blatter, N., Bergen, K., Nolte, O., Welte, W., Diederichs, K., Mayer, J., Wieland, M. and Marx, A.
• Detection of SARS-CoV-2 from raw patient samples by coupled temperature reverse transcription and amplification. PLoS ONE, 2020, 15(11), e0241740. Kuiper, J. W. P., Baade, T., Kremer, M., Kranaster, R., Irmisch, L., Schuchmann, M., Zander, J., Marx, A., Hauck, C. R.
• US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2. Emerging Infectious Disease, 2020, 26 (8), 1654-1665. Lu, X., Wang, L., Sakthivel, S.K., Whitaker, B., Murray, J., Kamili, S., Lynch, B., Malapati, L., Burke, S.A., Harcourt, J., Tamin, A., Thornburg,
  N. J., Villanueva, J.M., Lindstrom, S.

Material Safety Data

In accordance with OSHA 29CFR1910.1200, Australian [NOHSC:1005, 1008 (1999)] and EU Directives 67/548/EC, 1999/45/EC and 1272/2008 (CLP Regulation) this product may require a Material Safety Data Sheet (MSDS). This MSDS is available on request and on our webpage: https://www.mypols.de/products/volcano3g%C2%AE-direct-covid-19-kit
In general:
We recommend the use of gloves, lab coats and eye protection when working with these or other chemical reagents. myPOLS Biotec accepts no liability for any damage caused by handling or contact with this product.
This product is not of human, animal or plant origin. The product is obtained by recombinant protein expression in E. coli. The product is suitable for research use only and may be used for in vitro experiments.

Abbreviation Index

Amtsgericht Konstanz HRB 711269
St.-Nr.: 09048/20545
VAT No.: DE 294545185
Volcano3G® is a registered trademark of myPOLS Biotec GmbH.
www.mypols.de
© myPOLS Biotec GmbH, 2020

References

• myPOLS Biotec - Your specialist for DNA polymerases

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