PhoenixDx® 2019-nCoV RNA Detection Kit

Details for Product No. ABIN6952137
Antigen
Reactivity
SARS Coronavirus-2 (SARS-CoV-2)
2
Application
Quantitative real-time PCR (qPCR)
Options
Purpose Kit for qualitative RT-PCR-based detection of SARS-CoV-2
Brand PhoenixDx®
Sample Type Bronchoalveolar Lavage, Nasal Secretions, Oral Sample, Saliva, Tracheal aspirates
Characteristics PHOENIXDX® 2019-NCOV detects the presence of 2 different and highly specific gene sequences of SARS-CoV-2: E gene, and RdRP gene. Both assays must be tested positive to confirm the sample as SARS-CoV-2-positive.

Additionally, a non infectious positive control and a negative human extraction control are included. The positive control is used to confirm functionality of the assays and overall PCR performance, the negative human extraction control is included to evaluate the quantity of the RNA isolation independently from the 2019-nCoV assays.
Components
  • 50X VitaScript™ Reverse Transcriptase (100 μl)
  • PhoenixDx® 2X qPCR Mastermix E (500 μl)
  • PhoenixDx® 2X qPCR Mastermix RdRP (500 μl)
  • PhoenixDx® 2X qPCR Mastermix HEC (500 μl)
  • 2019-nCoV Target Positive Control (TPC) (50 μl)
  • Nuclease-free dH2O (2 x 1 ml)
Material not included
  • Suitable means & equipment for nucleic acid extraction (see chapter 3.4)
  • Real-time PCR detection system equipped for FAM™ detection
  • Adjustable pipettes & fitting filtered pipette tips
  • Appropriate PSA & workspaces for working with potentially infectious samples
  • Surface decontaminants such as DNAZap™ (Life Technologies), DNA Away™ (Fisher Scientific), RNAse Away™ (Fisher Scientific), 10 % bleach (1:10 dilution of commercial 5.25-6.0 % sodium hypochlorite)
  • Nuclease-free tubes / strips / plates to prepare dilutions, mastermixes etc.
  • Nuclease-free tubes / strips / plates corresponding to the PCR device
  • Suitable storage options for reagents and specimen (4 °C, -20 °C, -70 °C)
Target Name (Antigen)
Alternative Name Coronavirus (2019-nCoV)
Application Notes

Q: Can this kit be used as rapid-test?
No. This kit can only be used in fully equiped laboratories which have a RealTime PCR reader.

Q: Is the kit compatible with any Reader?
The kit is compatible with any Real-Time PCR Device able to detect FAM and HEX (and ROX, if required).

Q: Which specimens can be examined?
Respiratory specimens including nasopharyngeal / oropharyngeal aspirates or washes, nasopharyngeal oropharyngeal swabs, broncheoalveolar lavage, tracheal aspirates and sputum. Swab specimen s should be collected only on swabs with a synthetic tip (such as polyester or Dacron®) with aluminum or plastic shafts. Swabs with calcium alginate or cotton tips with wooden shafts are not acceptable.

Q: Additional equipment needed?

  1. Suitable means & equipment for nucleic acid extraction
  2. Real-time PCR detection system equipped for FAM™ detection
  3. Adjustable pipettes & fitting filtered pipette tips
  4. Appropriate PSA & workspaces for working with potentially infectious samples
  5. Surface decontaminants such as DNAZap™ (Life Technologies), DNA Away™ (Fisher Scientific), RNAse Away™ (Fisher Scientific), 10 % bleach (1:10 dilution of commercial 5.25-6.0 % sodium hypochlorite)
  6. Nuclease-free tubes / strips / plates to prepare dilutions, mastermixes etc.
  7. Nuclease-free tubes / strips / plates corresponding to the PCR device
  8. Suitable storage options for reagents and specimen (4 °C, -20 °C, -70 °C)

Q: What if dH2O controls (NTC) give a positive Ct for any assay?
If they do, the reaction was contaminated with sample RNA / DNA. Decontaminate equipment and workspace and repeat the reactions.

Q: If the HEC fails to amplify, must the sample still be considered positive?
For a sample to be considered positive for 2019 nCoV , both assays (E / RdRP) must give positive Ct values. If the HEC fails to amplify, the sample must still be considered positive.

Q: When is a sample to be considered negative for 2019-nCoV?
For a sample to be considered negative for 2019 nCoV , none of the 2 assays (E / RdRP) must give positive Ct. The HEC must give a positive Ct value (< 35 cycles) for these samples to ensure that sample material of suitable quality was present.

Q: What if Ct values for HEC are >35 cycles?
All reactions containing RNA isolate must give positive Ct values for the HEC assay. The Ct values should be < 35 cycles. Failure to amplify the negative human extraction control indicates a flawed RNA extraction or loss of RNA isolate due to RNAse contamination. The sample is not sufficient, results cannot be interpreted.

Q: What if negative Ct values for TPC are observed?
When using the TPC for 2019 nCoV , a positive Ct for both assays must be observed . The Ct value for the TPC should be < 35 cycles. If the Ct value does not correspond to the expected value or not all assays are tested positive , PCR was compromised . Check the reaction setup and PCR device settings and repeat the reactions. Repeated freeze and thaw cycles of the TPC can compromise its quality resulting in late Ct values.

Q: What if no amplification signal can be observed?
If no amplification signal is observed for any assay, PCR was inhibited. Check reaction setup and device settings and repeat the RNA extraction if necessary. Results are invalid and cannot be interpreted.

Q: What if only the E gene assay is observed to be positive?
If < 2 of the target assays are positive (e.g. only E gene, etc.), results are inconclusive. Check reaction setup and device settings and repeat the RNA extraction if necessary. Results are invalid and cannot be interpreted.

Q: I do not see a price for this kit. Instead it says "Contact our Customer Service for availability and price in your country."
Due to our supplier aggreement we are not allowed to sell this kit in Denmark, Turkey and UK. If you are located in another country please contact us and we will issue a quote for this kit.

Comment

Due to the intrinsic mutation rate of coronaviruses, it is possible that mutations in the target sequence occur and accumulate over time. This can lead to false-negative results with a PCRbased detection approach. PHOENIXDX® 2019-NCOV addresses this issue by using 2 detection assays on 2 different target sequences to minimize the chance of false-negative results caused by an altered target sequence.

If samples are tested negative in one or more assays, additional complementary testing may be required. The original target sequences for 2019-nCoV are included as a non-infectious target positive control (TPC) to check the integrity of the detection assays.

Samples tested positive should always be confirmed through complementary methods and additional analysis in an independent laboratory.

Assay Time 2 h
Protocol QPCR-BASED DETECTION OF 2019-NCOV
The first step in the detection of SARS-CoV-2 is the conversion of viral RNA into cDNA. Afterwards,the target sequences unique for SARS-CoV-2 are specifically amplified with amplification monitored in real time through the use of fluorescently labelled probes: upon incorporation into the newly amplified DNA strands, the fluorophore (FAM™) is released and an increase in fluorescence signal can be observed.
Reagent Preparation
  • Always prepare control reactions with nuclease-free dH2O instead of sample material (NTC) to detect contamination in your reagents.
  • Always include the assay for the negative human extraction control (HEC) to evaluate the quantity of your RNA isolate.
  • When using the provided target positive control (TPC), use 1 μL / reaction and add nuclease-free dH2O to 20 μL.
  • > 2 replicates / sample are strongly recommended.
  • Prepare enough mastermix for all planned reactions. It is recommended to prepare mastermix for 2 additional reactions to compensate for pipetting inaccuracies.
  • Distribute the mastermix to your strips/plate. An example setup is given in the images).
  • Prepare negative reactions first and seal them before handling positive samples. It is recommended to only bring potentially positive sample material and the included target positive control to the workspace once the NTC is prepared and sealed.
  • Add your samples to the Mastermix Plate. An example setup is given in the images).
  • Keep reactions on ice until transferring them to the PCR device.
Sample Collection Only use appropriate specimens for testing, such as:
  • Respiratory specimens including nasopharyngeal / oropharyngeal aspirates or washes,nasopharyngeal / oropharyngeal swabs, broncheoalveolar lavage, tracheal aspirates and sputum.
  • Swab specimens should be collected only on swabs with a synthetic tip (such as polyester or Dacron®) with aluminum or plastic shafts. Swabs with calcium alginate or cotton tips with wooden shafts are not acceptable

Handling and Storage
  • Specimens can be stored at 4 °C for up to 72 hours after collection.
  • If a delay in extraction is expected, store specimens at -70 °C or lower.
  • Extracted nucleic acids should be stored at -70 °C or lower.

Do not use specimens if
  • they were not kept at 2-4 °C (≤ 4 days) or frozen at -70 °C or below.
  • they are insufficiently labelled or lack documentation.they are not suitable for this purpose (see above for suitable sample material).the specimen volume is insufficient.
Sample Preparation
  • The performance of RT-PCR assays strongly depends on the amount and quality of sample template RNA. It is strongly recommended to qualify and validate RNA extraction procedures for recovery and purity before testing specimens.
  • Suitable nucleic acid extraction systems successfully used in combination with PHOENIXDX DETECTION KITS include: bioMérieux NucliSens® systems, QIAamp® Viral RNA Mini Kit, QIAamp® MinElute Virus Spin Kit or RNeasy® Mini Kit (QIAGEN), EZ1 DSP Virus Kit (QIAGEN), Roche MagNA Pure Compact RNA Isolation Kit, Roche MagNA Pure Compact Nucleic Acid Isolation Kit, and Roche MagNA Pure 96 DNA and Viral NA Small Volume Kit, and Invitrogen ChargeSwitch® Total RNA Cell Kit.
  • Store and keep residual specimens and extracted nucleic acids at -70 °C.
  • Only thaw the number of specimen extracts that will be tested in a single day.
  • Do not freeze/thaw extracts more than once before testing as each freeze/thaw cycle will decrease the RNA quality.
  • It may be possible to use patient samples directly, depending on the sample type. However, this may require a prior lysis step and titration of the amount on sample that can be used without inhibiting the reaction. This procedure has not been validated, use of isolated RNA is recommended.
Assay Procedure
  1. Make sure that all necessary equipment and devices are suitable, calibrated and functional before starting the experiments.
  2. Decontaminate equipment and workspace and prepare everything needed for the following experiment to keep the workflow short and repeatable.
  3. Switch on the PCR detection system and program it to avoid delays after setting up the reactions.
  4. Thaw all components of PHOENIXDX® 2019-NCOV on ice and mix gently but thoroughly to ensure even distribution of components. Collect liquid at the bottom of the tube with a quick spin.
  5. For first tests, prepare a dilution series of your RNA to determine your ideal concentration window. The recommended sample volume is <10 % of the final reaction volume.
  6. Set up your Mastermix Plate: (see images)
  7. Transfer the Mastermix Plate to a separate workspace to add the sample material. Preparing reagents and final reaction setup in separate workspaces helps to avoid contamination of equipment and reagents with sample material.
  8. Transfer the reactions to the PCR device, then cycle according to these guidelines: (see images)
  9. Once the run is finished, do not open the reaction tubes to avoid contamination and discard according to local guidelines and regulations. Do not autoclave as this may contaminate laboratory equipment with amplicons.
Calculation of Results
  • dH2O controls (NTC) must not give a positive Ct for any assay. If they do, the reaction was contaminated with sample RNA / DNA. Decontaminate equipment and workspace and repeat the reactions.
  • For a sample to be considered positive for 2019-nCoV, both assays (E / RdRP) must give positive Ct values. If the HEC fails to amplify, the sample must still be considered positive.
  • For a sample to be considered negative for 2019-nCoV, none of the 2 assays (E / RdRP) must give positive Ct values. The HEC must give a positive Ct value (< 35 cycles) for these samples to ensure that sample material of suitable quality was present.
  • All reactions containing RNA isolate must give positive Ct values for the HEC assay. The Ct values should be < 35 cycles. Failure to amplify the negative human extraction control indicates a flawed RNA extraction or loss of RNA isolate due to RNAse contamination. The sample is not sufficient, results cannot be interpreted.
  • When using the TPC for 2019-nCoV, a positive Ct for both assays must be observed. The Ct value for the TPC should be < 35 cycles. If the Ct value does not correspond to the expected value or not all assays are tested positive, PCR was compromised. Check the reaction setup and PCR device settings and repeat the reactions. Repeated freeze and thaw cycles of the TPC can compromise its quality resulting in late Ct values.
  • If no amplification signal is observed for any assay, PCR was inhibited. Check reaction setup and device settings and repeat the RNA extraction if necessary. Results are invalid and cannot be interpreted.
  • If < 3 of the target assays are positive (e.g. only E gene, only N gene etc.), results are inconclusive. Check reaction setup and device settings and repeat the RNA extraction if necessary. Results are invalid and cannot be interpreted.
Restrictions For Research Use only
Handling Advice Protect the 2X qPCR mastermixes from light as prolonged exposure can diminish the performance of the fluorophores.
Storage -20 °C
Storage Comment Store all components at -20°C and avoid repeated freeze and thaw cycles.
Supplier Images
Image no. 1 for PhoenixDx® 2019-nCoV RNA Detection Kit (ABIN6952137) Exemplary Result for RT-PCR
Image no. 2 for PhoenixDx® 2019-nCoV RNA Detection Kit (ABIN6952137) Pipetting Scheme for PCR
Image no. 3 for PhoenixDx® 2019-nCoV RNA Detection Kit (ABIN6952137) PCR setup ^1 Enable Data Collection for FAM™. If required, set Passive Reference t...
Image no. 4 for PhoenixDx® 2019-nCoV RNA Detection Kit (ABIN6952137) Example pipetting scheme for the distribution of mastermixes with the individual assa...
Image no. 5 for PhoenixDx® 2019-nCoV RNA Detection Kit (ABIN6952137) Example pipetting scheme for the addition of samples. The bottom half of the plate co...
Image no. 6 for PhoenixDx® 2019-nCoV RNA Detection Kit (ABIN6952137) Interpretation of amplification results with PhoenixDx 2019-nCoV RNA Detection Kit.