Advanced Molecular Diagnostics Zena Max SARS-CoV-2 Direct qPCR Detection Kit:
Product Information- RT PCR KIT Covid-19:
Catalogue Number KD145906D-100
RT PCR KIT Covid-19: Advanced Molecular Diagnostics Ltd. is a diagnostics company specializing in the manufacture and supply of molecular biology
instruments, reagents, and consumables.
Table of Contents:
- Intended Use
- Principles of the test
- Materials Provided
- Kit Contents
- Reagent Storage and Handling
- Materials and Equipment Required (not provided)
- RNA Extraction (if used)
- PCR Instrument
- Other Laboratory Equipment
- Warnings and Precautions
- Instrument compatibility
- Assay Procedure
- Sample Collection
- Acceptable specimen types
- PCR Set Up
- Data Analysis
- Interpretation of Results
- Performance Characteristics
- Clinical Performance
This assay is a Real-Time in vitro PCR test for the qualitative identification of the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2/COVID-19). It enables the direct amplification and identification of SARS-CoV-2 from human samples such as nasopharyngeal, nasal, oropharyngeal swabs.
It is based on the hydrolysis probe detection method and is a highly sensitive one-step RT-qPCR kit. With the help of different colored channels producing signals, the sample can be positive or negative.
For in vitro diagnostic use.
SARS-CoV-2 (COVID-19) is the virus responsible for respiratory disease caused by a novel (new) coronavirus that was first detected in Wuhan City, Hubei Province, China, later causing a global pandemic.
The virus is contagious and can be spread from humans to humans primarily through the exchange of mucus droplets that are expelled through sneezes or coughs.
The SARS-CoV-2 is a severe acute respiratory syndrome coronavirus. This outbreak is an important reminder that the global community must strengthen national and international programs for detection and response to future disease outbreaks.
Principles of the test:
Zena Max SARS-CoV-2 Direct qPCR assay system is a RealTime PCR that enables the direct amplification of COVID-19 infection in human samples like nasopharyngeal swabs (NPS), nasal swabs, nasal wash or aspirate or bronchoalveolar lavage (BAL) using one-step RT-qPCR (both reactions in the same tube) by the first reverse transcribing the genomic RNA target to cDNA, followed by amplification of the assay target and detection by the hydrolysis probe method of qPCR. The assay consists of a forward primer, a reverse primer, and a probe labelled with the 5’ FAM™ and ROX reporter dye with a 3’ quencher.
As the new target cDNA strand is synthesised, the tightly bound probe is cleaved by the 5’ to 3’ exonuclease activity of Taq polymerase which releases the fluorescent reporter from the quencher and substantially increases the fluorescent signal. The point at which the fluorescence becomes detectable.
above the background, the quantification cycle (Cq), is proportional to the amount of target present in the sample. The primers and probe sets target two different target region the S gene and N gene which had previously been used in the identification of the SARS coronavirus, however, there is no cross-reactivity with this or any other coronavirus sequenced thus far. The SARS CoV-2 (2019-nCoV) genomes are designed for the in vitro detection of SARS COV-2 genomes.
The lower the Cq, the greater the amount of target present. If, however SARS-CoV- 2 (COVID-19) is not present, a FAM signal or ROX signal will not be produced. An internal positive control assay is provided in order to assess the quality of the sample and the effect of any PCR inhibitors that may be present.
This assay contains two primers and a HEX labelled probe, designed to a highly conserved region of human RNA and a positive signal indicates that the RNA quality in the sample is acceptable for diagnostic testing. These assays are both incorporated into a ready-to-use PCR master mix that utilizes hot start technology, thus minimising non-specific reactions and ensuring maximum sensitivity.
*This in vitro diagnostic kit provides qualitative detection.
Figure 1. The principle of qPCR with hydrolysis probe detection for identifying the presence of ORF1 ab gene and N gene. The control assay in the master mix will produce a HEX signal if the sample quality is acceptable. The COVID-19 assay will produce a FAM & ROX signal, if COVID- 19 virus is present, however if it is not present, no FAM or ROX signal will be detected. Due to assay competition, the HEX signal may be reduced or absent when the other signals are strong.
Kit (RT PCR KIT Covid-19) Contents:
1 x 1.9 ml COVID-19 FAM, ROX and Human RNase P HEX multiplex qPCR master mix 1x 0.1ml RT Enzyme Mix
1 x 0.05 ml COVID-19 virus control
1 x 1 ml PCR Grade Water (nuclease free)
Reagent Storage and Handling:
- The RT PCR KIT Covid-19 should be transported and stored at temperatures between -30°C and -15°C.
- The kit will remain stable at least until the expiry date printed on the package, if the storage temperature is kept accordingly.
- Do not use the reagents beyond their expiration dates.
- Repeated freeze thawing of the kit components may result in lower detection quality.
- Avoid exposure to light.
- Ensure that all reagents are thoroughly thawed, mixed and pulse centrifuged before use.
- This is for in-vitro diagnostic use and for professional use only.
Materials and Equipment Required (not provided)
Universal Transport Media (UTM, Copan) or Universal Viral Transport (UVT, BD) to be used as a No Template Control (NTC).
RNA Extraction (if used): AMD recommends using the Luco-Viral NA Extraction Kit to extract DNA from the samples. Other leading kits, such as the QIAampMinElute Virus Spin Kit (Qiagen) or in-house methods are acceptable for use with this diagnostic kit, providing that it has been validated prior to use on patient samples.
PCR Instrument: This kit should be used with qPCR systems which can detect FAM and HEX fluorescent dyes. It is also compatible with both low and high ROX instruments.
Consumables: Use nuclease free PCR consumables appropriate to the qPCR instrument.
Other Laboratory Equipment: Vortex (for mixing samples), centrifuge (to collect components from the bottom of the tube), micro pipettes and sterile tips, tube rack, PCR tube/plate rack and spectrophotometer, disposable gloves, refrigerator (to store samples)
Warnings and Precautions: RT PCR KIT Covid-19
When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate safety data sheets (SDSs). Discard sample and assay waste according to your local safety regulations. It is essential to precisely follow the instructions in this manual, to ensure accurate results. Please familiarise yourself with this product manual and your qPCR instrument before using the AMD Zena Max SARS CoV-2 Direct qPCR Kit.
AMD Zena Max SARS-CoV-2 is compatible with the most common Real-Time PCR equipment as Biorad CFX96, Applied Biosystems 7500 Fast, QuantStudio 3,5,7, StepOne Plus, Aglient Mx3000, 3005P, Rotorgene Q, Cepheid Smartcycler, Analytik Jena qTower and Roche Lightcycler 480, 96.
The samples to be used with this kit(RT PCR KIT Covid-19) should be according to WHO recommendation which are the upper respiratory specimens like nasopharyngeal and oropharyngeal swab or wash in ambulatory patients or the lower respiratory specimens like sputum (if produced) and/or endotracheal aspirate or bronchoalveolar lavage in patients with more severe respiratory
disease. Please ensure that the sample is stored correctly and kept away from any contamination.
Acceptable specimen types:
Nasopharyngeal swabs or nasal swabs in Copan Universal Transport Media (UTM), BD Universal Viral Transport (UVT) or equivalent. Use only swabs with a synthetic tip (e.g. Dacron, nylon, or rayon) and an aluminum or plastic shaft. Do not use calcium alginate swabs, as they may contain substances that inhibit PCR testing.
Bronchoalveolar lavage (BAL) undiluted or diluted 1:1 (v/v) in a mucolytic.
- Nasal wash/aspirate undiluted.
1. Ensure that all reagents and samples are thawed completely, mixed, and briefly centrifuged. Keep all reagents and samples on ice during this procedure.
2. Set up the reactions using the table below.
- *Add directly to the PCR tubes/plate.
3. Add the RNA samples and the COVID-19 control to the PCR tubes/plate. Also add 5 l nuclease free water in place of the RNA as a No Template Control (NTC).
4. Seal the PCR tubes or plate and briefly spin to ensure that the reagents are at the bottom and no air bubbles are present.
5. Place the plate/tubes in the qPCR thermal cycler and use the following thermal profile:
- *Data collection step in FAM, ROX (diagnostic assay) and HEX (internal control assay) channels.
6. When the run has finished, dispose of the PCR reaction tubes/plate in an appropriate manner in accordance with local and national regulations.
Analyze the data if the software does not do this automatically at the end of the run. Export the data to Excel or a PDF report, depending on the qPCR instrument used, and view the results.
Interpretation of Results:
This is a qualitative assay and indicates the presence or absence of COVID-19 infection. The results should be interpreted as follows:
*Use Table.1 a quick reference guide
• The internal control assay signal in the HEX (yellow) channel should be present but may be absent or have a high Cq value (low signal) when the diagnostic assay signal is strong.• If there is a signal in the FAM (green) or ROX channel, with or without a HEX signal, the sample is positive for SARS-CoV-2 virus.
• If there is a HEX signal but no FAM or ROX signal, the sample is negative for SARS-CoV- 2 virus
• If there is no signal in either channels, the result is inconclusive.
- Table 1. Interpretation of the results obtained from the Zena Max SAR-CoV-2 virus qPCR Kit.
Quality: All AMD kits are manufactured under high quality standard methods and unique precision, compared with other leading commercial SARS-CoV-2 diagnostic kits.
Sensitivity: The Zena Max COVID-19 qPCR kit is highly sensitive, able to detect a minimum of 10 copies per reaction under our validation methods and devices.
Specificity: The analytical specificity study was carried out by in silico analysis shows that the AMD SARS-CoV-2 assays 100% sequence identity to 769 out of the 773 (99.4%) SARS- CoV-2 genomes available in the public databases, however, there is no cross-reactivity with any other coronavirus sequenced or the most common respiratory pathogens thus far.
The clinical performance of Zena Max SARS-CoV-2 Real Time PCR Detection kit was tested using clinical specimens in transport medium. 174 individual negative nasopharyngeal specimens and a total of 18 positive clinical samples tested with a validated molecular comparator assay.
Low positive contrived clinical samples were prepared by spiking a quantified clinical sample into individual negative clinical samples to approximately 30 samples.
All clinical samples were collected from patients with signs and symptoms of upper respiratory infection by qualified personnel according to the package insert of the collection device and stored frozen until use.
These results were compared with those obtained with a molecular detection method. The results were as follows:
RT PCR KIT Covid-19: This kit is for in vitro diagnostic procedures and should only be used by specifically trained laboratory personnel. The expiry date of all components must be checked before use and disposed of if expired. Occasionally mutations may arise in the region of the genome targeted by the primers and probes of this assay, leading to reduction in performance or failure of the assay. The assay design and efficacy is reviewed periodically.
This assay can be used with other types of samples that have been validated only with RNA extracted from respiratory samples (nasopharyngeal swab and oropharyngeal swab).
False Negative results may arise from several factors and their combinations including improper specimens as “collection, storage, and transportation methods” or degradation of the viral RNA during sample shipping and storage. The presence of RT-qPCR inhibitors or other types of interfering substances. The impacts of vaccines, antiviral therapeutics, antibiotics, chemotherapeutics or immunosuppressant drugs used to prevent COVID-19 infection or used during the treatment of the infection have not been evaluated.
AMD produces real-time PCR kits with a wide range of applications for researchers from gene expression analysis, cDNA and population genotyping studies to the multiplex detection of several disease targets real-time PCR with excellent sensitivity and specificity.
*This is a Direct kit which can also work with RNA extraction
If you have any queries, comments, or complaints please refer to our website at www.am-diagnostics.co.uk
Also Check out: COVID-19 division