Automated wash and reuse of disposable pipette tips in a SARS-CoV-2 RT-qPCR diagnostic pipeline.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic led to global shortages in laboratory consumables, in particular for automated PCR. The Technical University of Denmark supported Danish hospitals from 2020 to 2022, conducting SARS-CoV-2 RT-qPCR on around 10,000 patient samples daily. We encountered shortages of disposable pipette tips used with automated liquid handlers that transferred oropharyngeal swab samples to 96-well microplates before RNA extraction. To enable tip reuse, we developed an automated protocol for washing tips with a 0.5 % sodium hypochlorite solution. This effectively eliminated carry-over of genomic material and the wash solution remained effective when stored in an open reservoir at ambient temperatures for 24 h. A three-day validation setup demonstrated the robustness of the tip wash protocol. Reducing the number of tips used for transferring samples to 96-well microplates from 96 to 8 enabled us to mitigate pipette tip shortages, lower costs, and minimize plastic waste generation.

SpikeSeq: A rapid, cost efficient and simple method to identify SARS-CoV-2 variants of concern by Sanger sequencing part of the spike protein gene.

In 2020, the novel coronavirus, SARS-CoV-2, caused a pandemic, which is still raging at the time of writing this. Here, we present results from SpikeSeq, the first published Sanger sequencing-based method for the detection of Variants of Concern (VOC) and key mutations, using a 1 kb amplicon from the recognized ARTIC Network primers. The proposed setup relies entirely on materials and methods already in use in diagnostic RT-qPCR labs and on existing commercial infrastructure offering sequencing services. For data analysis, we provide an automated, open source, and browser-based mutation calling software (https://github.com/kblin/covid-spike-classification, https://ssi.biolib.com/covid-spike-classification). We validated the setup on 195 SARS-CoV-2 positive samples, and we were able to profile 85% of RT-qPCR positive samples, where the last 15% largely stemmed from samples with low viral count. We compared the SpikeSeq results to WGS results. SpikeSeq has been used as the primary variant identification tool on > 10.000 SARS-CoV-2 positive clinical samples during 2021. At approximately 4€ per sample in material cost, minimal hands-on time, little data handling, and a short turnaround time, the setup is simple enough to be implemented in any SARS-CoV-2 RT-qPCR diagnostic lab. Our protocol provides results that can be used to choose antibodies in a clinical setting and for the tracking and surveillance of all positive samples for new variants and known ones such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) Delta (B.1.617.2), Omicron BA.1(B.1.1.529), BA.2, BA.4/5, BA.2.75.x, and many more, as of October 2022.