PhoenixDx® SARS-CoV-2 IVD

Produktnummer ABIN6952545

Produktdetails

Target: SARS-CoV-2 (SARS-Coronavirus-2 (SARS-CoV-2))

Reaktivität: SARS Coronavirus-2 (SARS-CoV-2)

Applikation: Quantitative real-time PCR (qPCR)

Verwendungszweck: PHOENIXDX® SARS-COV-2 IVD is a real-time RT-PCR-based diagnostic test for the in vitro qualitative detection of 2019-Novel Coronavirus (SARS-CoV-2) in respiratory specimens and sera from patients who meet COVID-19 clinical and/or epidemiological criteria. It is intended for use by clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures. The kit follows CDC’s and WHO’s latest detection guidelines (03/2020).

Marke: PhoenixDx®

Proben: Bronchoalveolar Lavage, Nasal Secretions, Oral Sample, Saliva, Tracheal aspirates

Produktmerkmale: PHOENIXDX® SARS-COV-2 IVD is a real-time RT-PCR-based detection system for the 2019 Wuhan coronavirus (SARS-CoV-2, formerly 2019-nCoV). SARS-CoV-2 is considered a novel human coronavirus that is genetically distinct from the common human coronaviruses (229E, NL63, OC43, HKU1), which cause seasonal acute respiratory illness. It is also genetically distinct from the two newer human coronaviruses, MERS-CoV and SARS-CoV.

PHOENIXDX® SARS-COV-2 IVD detects the presence of 2 different and highly specific gene sequences of corona viruses: one identifies SARS-CoV-2, SARS-CoV, and bat-SARS-related coronaviruses (Sarbeco), one specifically targets SARS-CoV-2 (SARS-CoV-2). Additionally, a non-infectious target positive control (TPC) and a negative human extraction control (HEC) 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 quality of the RNA isolation independently from the SARS-CoV-2 assays.

Bestandteile:

• PhoenixDx® Enzyme Mix (1 x 150 μl)
• PhoenixDx® Sarbeco Mix (1 x 750 μl)
• PhoenixDx® CoV-2 Mix (1 x 750 μl)
• PhoenixDx® HEC Mix (1 x 750 μl)
• SARS-CoV-2 TPC (1 x 200 μl)

Benötigtes Material:

• 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
• Nuclease-free water
• 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)

Anwendungsinformationen

Applikationshinweise: QPCR-BASED DETECTION OF SARS-COV-2
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.
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® SARS-COV-2 IVD 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. The original target sequences for SARS-CoV-2 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.
PHOENIXDX® SARS-COV-2 IVD is validated on the 7500 Fast System (ABI®).

Kommentare:

PHOENIXDX® SARS-COV-2 IVD detects SARS-CoV-2 RNA in nasopharyngeal and oropharyngeal swab samples during infection. Positive results indicate the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information must be considered to determine the actual patient infection status. Positive results do not exclude bacterial infection or co-infection with other viruses.
Negative results do not exclude a SARS-CoV-2 infection and must not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.
The use of PHOENIXDX® SARS-COV-2 IVD is intended for use by clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures.
The kits follow CDC’s and WHO’s latest detection guidelines (03/2020).

Aufbereitung der Reagenzien:

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® SARS-COV-2 IVD 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. Set up your Mastermix Plate:
a. Always prepare control reactions with nuclease-free dH2O instead of sample material (NTC) to detect contamination in your reagents.
b. Always include the assay for the negative human extraction control (HEC) to evaluate the quality of your RNA isolate.
c. When using the provided target positive control (TPC), use 4 μl / reaction.
d. > 2 replicates / sample are strongly recommended.
e. Prepare enough mastermix for all planned reactions. It is recommended to prepare mastermix for 2 additional reactions to compensate for pipetting inaccuracies.
f. Distribute the mastermix to your strips/plate.
6. 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.
   a. 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.
   b. Add your samples to the Mastermix Plate.
   c. Keep reactions on ice until transferring them to the PCR device.
7. Transfer the reactions to the PCR device, then cycle according to the guidelines.
8. 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.

Probennahme: 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 recommended as they may contain substances that inactivate some viruses and inhibit PCR testing and should only be used if dacron or rayon swabs are not available.

- 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.
- Clinical specimens must be considered potentially infectious and treated accordingly.
- Do not vortex specimens as this will fragment the RNA and lead to failure of the PHOENIXDX® SARS-COV-2 IVD assays.

Aufbereitung der Proben:

• 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: Quick-RNA Viral Kits (Zymo Research), 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.
• Only extract the number of specimens 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. For optimal results, use directly and do not freeze and thaw before use.
• Extracted nucleic acids should be stored at -70°C or lower and (if re-testing is expected) stored in aliquots.

Ergebnisberechnung:

• dH2O controls (NTC) must not give a positive Ct for any assay. If they do, the reaction was contaminated with sample RNA / cDNA. Decontaminate equipment and workspace and repeat the reactions. If the contamination persists, use fresh reagents.
• For a sample to be considered positive for SARS-CoV-2, the assay for SARS-CoV-2 and/or Sarbeco must give a positive Ct value. Amplification of the HEC is expected around Ct 22-29. Should the HEC fail to amplify, the sample must still be considered positive. An additional positive signal for Sarbeco confirms the result but is not obligatory.
• For a sample to be considered negative for SARS-COV-2, the SARS-CoV-2 and Sarbeco assay must not give a positive Ct value. The HEC must give a positive Ct value (Ct 22-29) for these samples to ensure that sample material of suitable quality was present. If the assay for Sarbeco gives a positive Ct value, is considered presumptive positive for SARS-CoV-2. A negative SARS-CoV-2 result and a positive Sarbeco result is suggestive of low concentration of viral RNA, a mutation in the SARS-CoV-2 target sequence, or an infection with other Sarbecovirus (e.g., SARS-CoV or some other Sarbecovirus previously unknown to infect humans).
• A sample is considered negative for any of the tested corona viruses if the assays for Sarbeco and SARS-CoV-2 do not give a positive Ct value while the HEC still amplifies with the expected Ct value (Ct 22-29).
• 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.
• All reactions containing RNA isolate must give positive Ct values for the HEC assay. The Ct values are expected around 22-29. Failure to amplify the negative human extraction control indicates a flawed RNA extraction or loss of RNA isolate due to RNAse contamination. Late Ct values for the HEC may indicate a low RNA quality / amount in the extract.
• When using the TPC for SARS-COV-2, a positive Ct for both assays must be observed. The Ct value for the TPC should be < 35 cycles. The HEC must not give a signal when using the TPC. 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.

Always analyze your sample reactions independently of the TPC reactions. The TPC is an artificial control construct resulting in a significantly higher signal strength than actual samples. This will lead to a distorted picture when analyzed together with actual samples.
For analysis, the threshold must be set only for the wells containing sample material not including wells with TPC reactions. If amplification in sample reactions seems to have failed, check if the TPC reactions are displayed simultaneously.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: -20 °C

Informationen zur Lagerung: Store all components at -20°C and avoid repeated freeze and thaw cycles. Protect the 2X qPCR mastermixes from light as prolonged exposure can diminish the performance of the fluorophores.

Antigendetails

Target: SARS-CoV-2 (SARS-Coronavirus-2 (SARS-CoV-2))

Andere Bezeichnung: SARS-Coronavirus-2 (SARS-CoV-2 Produkte)

Substanzklasse: Virus