Rapid detection of SARS-CoV-2 by pulse-controlled amplification (PCA)

Abstract

In the current pandemic of SARS-CoV-2, rapid identification of infected individuals is crucial for management and control of the outbreak. However, transport of samples, sample processing and RT-qPCR analysis in laboratories are time-consuming. Here we present a prototype of a novel nucleic acid-based test format - pulse controlled amplification - that allows detection of SARS-CoV-2 directly from up to eight swab samples simultaneously without the need for RNA extraction within 25 min with a sensitivity of 100 % for samples with a viral load of ≥ 1.6 × 10e3 copies/μl This new principle might pave the way to rapid and sensitive point of care testing.

Keywords: COVID-19; Diagnostics; Point of care; Rapid test; SARS-CoV-2.

Fig. 1

PCA performed with A) a dilution series of virus culture B) clinical samples. Dashed line denotes threshold fluorescence. The dilution series was performed 3 times. The curves are representatives of one experiment. For the clinical samples the curves are representatives of the 83 clinical samples tested.

Fig. 2

Probit analysis was done in replicates of eight using SARS-CoV-2 inactivated with 4 volumes of AVL buffer. Viral RNA was immobilized on the wires in two rounds of bind/wash. The limit of detection is 4.9 copies/μl (3.8 – 8.2 copies/μl, 95 % confidence interval). Extrapolated to the target copy number per PCA reaction, this equates to 443.7 copies (341.1 – 738.0 copies, 95 % confidence interval). Dashed black lines denote the 95 % confidence interval.

Fig. 3

Comparison of the performance of PCA and standard RT-qPCR using clinical sample material (swab samples in universal transport medium). The figure includes 74 out of 83 tested samples. The remaining 9 samples were positive with RT-qPCR but negative with PCA.