Processing Hundreds of SARS-CoV-2 Samples with an In-House PCR-Based Method without Robotics

Theresa Mair,Helmut Salzer,Angelika Heissl,Christian Paar,Bernd Lamprecht,Elisabeth Schreier-Lechner,Maja Ivankovic,Irene Tiemann-Boege

doi:10.3390/v13091712

Theresa Mair, Helmut Salzer + Show 6 more

Open Access

https://doi.org/10.3390/v13091712

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Abstract

The SARS-CoV-2 pandemic has required the development of multiple testing systems to monitor and control the viral infection. Here, we developed a PCR test to screen COVID-19 infections that can process up to ~180 samples per day without the requirement of robotics. For this purpose, we implemented the use of multichannel pipettes and plate magnetics for the RNA extraction step and combined the reverse transcription with the qPCR within one step. We tested the performance of two RT-qPCR kits as well as different sampling buffers and showed that samples taken in NaCl or PBS are stable and compatible with different COVID-19 testing systems. Finally, we designed a new internal control based on the human RNase P gene that does not require a DNA digestion step. Our protocol is easy to handle and reaches the sensitivity and accuracy of the standardized diagnostic protocols used in the clinic to detect COVID-19 infections.

Highlights

In late 2019 and early 2020, the first cases with viral pneumonia of unknown cause were reported in Wuhan, China, which were later identified as being caused by the novel SARS-CoV-2 virus [1,2], and has since caused a severe global pandemic
The principle of our in-house open-system PCR-based assay to detect SARS-CoV-2 viral RNA was adapted from the process of the Drosten group [11,12] and Centers for Disease Control and Prevention (CDC)
Extracted RNA was transcribed into cDNA by a reverse transcriptase (RT) followed by amplification of specific regions with quantitative real-time PCR in a one-step reaction (RT-quantitative PCR (qPCR)) using primer/probe sets (TaqMan-based chemistry) that recognizes the viral genome

Summary

Introduction

In late 2019 and early 2020, the first cases with viral pneumonia of unknown cause were reported in Wuhan, China, which were later identified as being caused by the novel SARS-CoV-2 virus (severe acute respiratory syndrome coronavirus 2) [1,2], and has since caused a severe global pandemic. The high reproductive rate of SARS-CoV-2 resulted in a world-wide spread of the virus that is being contained by social isolation, vaccination, and extensive testing. Many testing strategies have been developed that use nasal or throat swabs Those tests are based on the detection of the viral RNA (PCR-based tests) and other cheap, fast virus tests that measure viral antigen or antibodies and return a result within 15 min on a paper strip. These latter tests are a qualitative measure of infection or no-infection and detect only very high viral loads. PCR-based tests are still considered the golden standard to detect SARS-CoV-2 and will stay as the main test for screening the pandemic [8]

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