Monitoring with self-sampling and PCR
COVID-19 RT-qPCR testing pipeline at the Vienna BioCenter
On 20 April 2020, the research institutes at the Vienna BioCenter started testing their employees for SARS-CoV-2 twice weekly on a voluntary basis. The initiative was launched by researchers at the IMP and IMBA, with dozens of scientists, technicians and administrative staff at the Vienna BioCenter involved in developing, setting up and running the assay.
Under the “Vienna COVID-19 Detection Initiative” (VCDI) and with support from the Vienna Science and Technology Fund (WWTF), the project was scaled up and extended to offer testing to university departments, companies, schools, nursing and retirement homes. By March 2021, roughly 88,000 samples had been analysed and 84 positive cases were detected. Comprehensive testing and contact tracing have turned the Vienna BioCenter into a safe space for its employees and allowed research activities to continue.
(1) Samples for coronavirus-testing are obtained by gargling with HBSS buffer. This simple, non-invasive procedure was implemented and optimised by researchers at the Vienna BioCenter and yields samples of consistently good quality.
(2) The test kit consists of a 50 ml Falcon tube, a 1 ml data matrix tube and a disposable pipette. Gargling is performed at home, following a detailed protocol. A small amount of the throat-wash liquid (0.75 ml) is pipetted into the data matrix tube which is dropped off at the submission fridge.
(3) Every morning, the matrix racks containing the code-labelled tubes are picked up by a member of the SARS-CoV-2 screening team and taken to the testing lab, a unit of the Vienna BioCenter Core Facilities which is equipped and staffed for automated sample processing.
(4) More tubes arrive by courier from schools and university departments. On a typical day, 10 to 15 racks are processed, each containing up to 96 sample tubes. The facility is able to process different types of data matrix tubes, allowing for greater flexibility.
(5) Within the racks, sample tubes are arranged in an 8x12 pattern, compatible with the standard 96-well format widely used in labs. 2D-barcodes at the bottom of the tubes are used to identify and trace each sample.
(6) A scanner reads the 2D-barcodes and feeds the information into a database. From now on, each sample is automatically identifiable and traceable throughout the entire process.
(7) The following steps are performed under sterile conditions in a laminar flow hood. The de-capper automatically removes the screw caps from the matrix tubes and later re-applies them. It goes through an entire rack in just thirty seconds. Different cartridges are available for different tube models and can be rapidly adjusted.
(8) The tubes are now open, and a pipet robot (Integra VIAFLO) adds DTT which rapidly dissolves mucus and reduces the viscosity of the samples. After a short spin on the centrifuge, the pipet robot removes the supernatant and the remaining sediment containing the biological material is used for sample pooling.
(9) Up to 10 samples can be pooled. This strategy allows for higher throughput while still retaining the necessary sensitivity. The samples are incubated with lysis buffer to break up the cells and inactivate the SARS-CoV-2 virus.
(10) Magnetic beads are added to the samples and subsequently placed in the KingFisher Flex where RNA preparation takes place. It takes the KingFisher robot 30 minutes to obtain pure RNA from the sample pool. The automated RNA extraction method is based on carboxylated magnetic bead separation.
(11) The samples are prepared for RT-qPCR by adding the RNA to the mastermix. The mastermix contains primers for viral and human RNA as well as probes with up to five different fluorophores, boosting the throughput and sensitivity even further.
(12) The plates are now moved to the thermo-cyclers. This is where RT-qPCR, the process at the heart of SARS-CoV-2 detection, takes place. Up to 45 cycles, each doubling the amount of viral RNA, are carried out. If a positive signal is detected, the respective pool is opened up and back-up samples are re-tested individually.
(13) In the cold-room, back-ups of samples are stored for re-testing or in case a pool has to be opened up. Except for positive samples, the back-ups are discarded after one week.
(14) Once the RT-qPCR assays are completed, the data are analysed and the results are imported into a database. The web-based COVID-19 testing database solution was developed in-house at the VBCF. It supports sample registration and tracking through all steps of the experimental pipeline, storage and approval of all test results, and an anonymous user administration.
(15) Within the same day, users obtain their test results anonymously by logging onto a website and entering their unique sample ID. The communicated results include the Cq values, an interpretation of the test result and sample quality, and optional comments.
Acknowledgements & credit
The ongoing testing-initiative was made possible by a joint effort of the Vienna BioCenter community. Development of the testing pipeline was led by Johannes Zuber, who together with Harald Scheuch, Robert Heinen, Marcus Dekens and many volunteers established all protocols and automated workflows. During the first months, tests were carried out and further optimised by the Molecular Biology Service of IMP, IMBA and GMI with great help from volunteers from numerous research groups. Johanna Trupke and her team took over in the fall of 2020. The database application at the core of administering the tests was developed by Thomas Micheler and the Strategic Information Management team at VBCF. We are also grateful to our colleagues in administration who chase up supplies, deal out the kits and keep us informed, and to our management for making it all possible.
Image 1: IMP, Images 2-15: L. Schedl / IMP.