Victor Chen1, Kiranmai Durvasula2
1. Chroma ATE, Inc, Guishan Dist., Taoyuan City, Taiwan 333001
2. Omega Bio-tek, Inc, Norcross, GA 30071
Introduction
In response to the COVID-19 pandemic, the demands for nucleic acid purification and laboratory automation have increased dramatically. The Mag-Bind® Universal Pathogen 96 Kit (M4029) was developed by Omega Bio-tek to provide an automated solution for the purification of viral nucleic acids with pathogenic DNA from a range of samples. In this application note, we provide the automated workflow for viral RNA extraction and PCR set up for detection of SARS-CoV-2 RNA using the Mag-Bind Universal Pathogen 96 Kit on Chroma ATE’s MagXtract 3200. The product specifications of Chroma ATE’s MagXtract 3200 system are described in Table 1 below. The application note also discusses the performance of this automated workflow in terms of sensitivity of viral detection using real-time RT-PCR.
Table 1. Product specifications of Chroma ATE’s MagXtract 3200 system.
Materials and Methods
Inactivated SARS-CoV-2 from Amplirun® Total SARS-CoV-2 Control (Swab) (Vircell Microbiologists) was used as the source for viral RNA extraction. 20-fold and 40-fold serial dilutions of inactivated SARS-CoV-2 was purified using the Mag-Bind Universal Pathogen 96 Kit, following the manufacturer recommended procedure on the MagXtract 3200 instrument. Briefly, 250 μL of the serial dilutions of the viral samples were thoroughly mixed with 275 μL SLX-Mlus Buffer. The initial lysis and homogenization steps were performed manually, offline. The remaining steps in the Kit’s protocol: bind, wash, dry, and elute, in addition to the qRT-PCR setup, were fully automated on the MagXtract 3200. The liquid handler performs all bead collection, transfer, and dispense operations as necessary. The layout of the extraction workflow in a 96-well deep-well plate is as described in Figure 1.
Extracted viral RNA was analyzed using a qRT-PCR kit from Vircell microbiologists on the LightCycler® 96 from Roche.
Figure 1. Extraction layout of the buffers in a 96-well deep-well plate on MagXtract 3200 platform.
MagXtract 3200 Open System and Automation Solution
The MagXtract 3200 allows the authorized user to edit its protocols, including nucleic acid extraction and PCR preparation. The MagXtract 3200 is highly flexible and can easily fit into the workflows of most magnetic bead-based extraction kits and PCR reagent kits.
Open System
Following manufacturer’s protocols, the automated steps on MagXtract 3200 can be optimized to achieve excellent performance.
Automation Solution
Automation of the magnetic bead-based system reduces the number of manual steps required for extraction and PCR preparation.
GUI
The MagXtract 3200 has three operating modes: full run, preparation, and extraction.
MagXtract 3200 software provides protocol-based control to streamline workflows. The stepwise GUI and touchscreen control guide the user through the completion of the assay setup, from sample loading to the consumable placement.
Results and Discussion
The sensitivity of the Mag-Bind Universal Pathogen 96 Kit was evaluated with a qRT-PCR reaction. Figure 2 shows the amplification curves of the extracted viral RNA as detected using a qPCR kit from Vircell on the LightCycler 96 from Roche. According to the manufacturer specifications, Amplirun total SARS-CoV-2 Control (Swab) at 20-fold dilution is equivalent to 1500 copies/mL (9.36 copies/qPCR vial) and at 40-fold dilution, it is equivalent to 750 copies/mL (4.68 copies/qPCR vial). The amplification curves at 20-fold and 40-fold dilutions of viral RNA are illustrated in Figure 2. The discernible exponential region at even 40-fold dilution suggests high sensitivity of SARS-CoV-2 detection using Mag-Bind Universal Pathogen 96 Kit (M4029) at viral loads as low as 4.68 copies/qPCR vial.
High Sensitivity of Viral RNA Detection at Low Viral Loads
Figure 2. Amplification curves of 20-fold and 40-fold dilutions of inactivated SARS-CoV-2 virus extracted using Omega Bio-tek’s Mag-Bind Universal Pathogen 96 Kit on the MagXtract 3200.
Conclusions
Omega Bio-tek’s Mag-Bind Universal Pathogen 96 Kit in conjunction with Chroma ATE’s MagXtract 3200 for viral RNA extraction presents a solution to the increasing demand for timely high throughput viral RNA purifications by streamlining the workflow for extraction and qPCR setup. The Mag-Bind Universal Pathogen 96 Kit has been shown to yield viral RNA which can be detected even at a low viral load, making it useful for detecting viruses such as SARS-CoV-2 via RT-PCR. This workflow can potentially save 80% of the hands-on time and provide researchers and scientists to conduct clinical work in a safe, accurate, and efficient manner.
Product Information
WP-0040