Established in Shanghai in 2010, 3D Medicines (3DMed) is one of the first companies to provide oncology precision medical testing services in China. Over the years, the Company has grown into a successful Group consisting of 3DMed Diagnostics (Full company name: Etis Biotechnology (Shanghai) Co., Ltd.) .With the former already well-established as a market leader in automated and smart precision diagnostics in China.
The Company works with more than 800 hospitals, to provide them with products and services that cover the entire process of precision medicine from early disease diagnosis, companion diagnostics to dynamic monitoring.
Guided by its “Patients First” motto, the Company stepped up and joined the fight against the pandemic immediately after the outbreak of the novel coronavirus in 2020. Due to its significant contributions, the Company, based in Minhang district was recognized as a “key organization in the fight against COVID19 pandemic (Shanghai)”. At the same time, the Company has assisted in the global fight against the pandemic by developing related products and solutions such as novel coronavirus nucleic acid test kit, automated nucleic acid extraction kit, and mobile testing shelters. Its products and services are available in more than 60 countries and regions including Europe, U.S., Brazil, Australia, Middle East, Southeast Asia, and Africa.
Results are the identification of SARS-CoV-2 RNA. The RNA is generally detected in oropharyngeal/nasopharyngeal swab and saliva specimens during infection. Positive results are indicative of active infection with SARS-CoV-2.
The test consists of three processes in a single tube assay:
• Reverse transcription of target RNA and Internal Control RNA to cDNA
• PCR amplification of target and Internal Control cDNA
• Simultaneous detection of PCR amplicons by fluorescent dye labelled probes
The diagnostic kit contains:
➢ RT-PCR Reaction Mix (850 μl/tube) composed of 3 sets of primers and probe designed to detect the Sars Cov 2 RNA:
• ORF1 ab that is detected with FAM fluorophore
• E gene that is detected by VIC fluorophore
• N gene that is detected by ROX fluorophore
and 1 set of primers and probes designed to detect the internal control:
• Rnase-P that is labeled with Cy5 fluorophore.
➢ Enzyme mix (150 μL/tube) composed of Taq polymerase, Reverse transcriptase and UDG (Uracil DNA glycosylase)
➢ Positive control (1000 μL/tube)
➢ Negative control (1000 μL/tube)
ANDiS FAST SARS-CoV-2 RT-qPCR Detection Kit shall be stored at -25℃ to -15℃. It is important to protect SARS-CoV-2 Assay which contains fluorogenic probes from light.
Reagent preparation
All the reagents and controls need to be equilibrated at room temperature, except for the enzyme. The Enzyme Mix has to be kept in cooler or on ice during the preparation and use. All the reagents and controls are vortexed for 10 seconds, centrifuged briefly to collect the content to the bottom of the tube.
Preparation of RT-qPCR Mix according to the formula described in the Table below:
Reagent Name | Volume in μL per Reaction | Volume in μL per N Reactions |
|---|---|---|
| RT-qPCR Reaction Mix | 8.5 | 8.5 × (N+1) |
| Enzyme Mix | 1.5 | 1.5 × (N+1) |
| Total Volume | 10 | 10 × (N+1) |
After the mix is prepared, add 10μL of RT-qPCR Mix into each required well of an appropriate optical 96-well reaction plate and 10μL of extracted RNA of samples, Positive Control and Negative Control into each well. Seal the optical 96-well reaction plate with optical adhesive file, spin the optical 96-well reaction plate in order to avoid drops on the foil and collect the entire content to the bottom of the wells.
RT-qPCR amplification
In Biorad CFX-96 the general settings to define are:
• Sample Volume: 20μL
• The Fluorescent Detectors (Dye) that are four (FAM, VIC, ROX, CY5)
• The Thermal Cycle profile: “File-Open-New protocol”
Stage | Temperature | Time | Cycle Number |
|---|---|---|---|
| 1 | 50° | 2 minutes | 1 |
| 2 | 95° | 2 seconds | 1 |
| 3 | 95° | 1 second | 41 |
| 60° | 13 seconds |
Fluorescent signal is collected at 60℃ step.
Analyzing the data using Biorad CFX-96 software:
• Select FAM, VIC, ROX and CY5 to setting the Baseline Threshold, respectively. Unselect the box for “Auto Calculated”.
• For Single threshold setting: manually drag the threshold line until it lies within the exponential phase of the fluorescence curve and above any background signal.
• Determine the cycle threshold (Ct) values for each assay.
Interpretation of results
Specimen ID | ORF1 ab (FAM) | N gene (ROX) | E gene (VIC) | Internal Control (Cy5) | Results | Interpretation |
|---|---|---|---|---|---|---|
| A | + | + | + | +/- | SARS-CoV2 positive | SARS-COV2 RNA is detected |
| B | + | - | +/- | +/- | SARS-CoV2 positive | SARS-COV2 RNA is detected |
| C | - | + | +/- | +/- | SARS-CoV2 positive | SARS-COV2 RNA is detected |
| D | + | + | +/- | +/- | SARS-CoV2 positive | SARS-COV2 RNA is detected |
| E | - | - | + | +/- | Presumptive positive for SARS-COV2 | Sarbecovirus RNA is detected but not specific SARS-COV2 |
| F | - | - | - | + | SARS-COV2 negative | SARS-COV2 RNA is not detected |
| G | - | - | - | - | Invalid | Repeat the test |
“+” means “Detected”, and the Ct value ≤ 40. And “-” means “Not Detected”, and the Ct value > 40.
E gene could not distinguish between SARS-CoV-2 and SARS-CoV. Additional confirmatory testing may be conducted if it is necessary to differentiate between SARS-CoV-2 and SARS-CoV or other Sarbecovirus currently unknown to infect humans.
The analytical sensitivity of this kit is 200 copies/ml. The kit is reliable because is possible in one step detect N, E and ORF1 ab genes of SARS-CoV-2 RNA that indicate the current infection and these results are obtained in a short time (around 40 minutes).
This kit meets safety, quality and performance requirements.