Microchip RT-PCR Detection of Nasopharyngeal SARS-CoV-2 Samples

Abstract

Fast, accurate, and reliable diagnostic tests are critical for controlling the spread of the coronavirus disease 2019 (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The current gold standard for testing is real-time PCR; however, during the current pandemic, supplies of testing kits and reagents have been limited. We report the validation of a rapid (30 minutes), user-friendly, and accurate microchip real-time PCR assay for detection of SARS-CoV-2 from nasopharyngeal swab RNA extracts. Microchips preloaded with COVID-19 primers and probes for the N gene accommodate 1.2-μL reaction volumes, lowering the required reagents by 10-fold compared with tube-based real-time PCR. We validated our assay using contrived reference samples and 21 clinical samples from patients in Canada, determining a limit of detection of 1 copy per reaction. The microchip real-time PCR provides a significantly lower resource alternative to the Centers for Disease Control and Prevention-approved real-time RT-PCR assays with comparable sensitivity, showing 100% positive and negative predictive agreement of clinical samples.

Figure 1

A: AriaDNA analyzer. B: Microchip for coronavirus disease 2019 detection with lyophilized reagents in the microwells displayed along with its packaging. C: Layout of the microchip. Each sample and its associated control reactions are loaded into three wells, nCoVN1 (novel coronavirus N1 primer/probe], nCoVN2 (novel coronavirus N2 primer/probe), and Homo sapiens ribonuclease P/MRP subunit p30 (HsRPP30) (human sample control primer/probe) at 1.2 μL each. Three controls are loaded per chip. NEC, negative extraction control; NTC, negative template control; PTC, positive template control.

Figure 2

Limit of detection (LoD) determination with extracted cultured severe acute respiratory syndrome coronavirus 2 RNA. Ct values of 10-fold and 2-fold serial dilutions of extracted cultured viral RNA obtained by microchip quantitative RT-PCR assay and measured on the AriaDNA analyzer with N1 (green dots) (A) and N2 (blue dots) (B) primers/probes. Ct values of 20 positive replicates per concentration of viral RNA (1 copy per well, 0.72 copies per well, and 0.25 copies per well) and 12 double distilled water negative controls per viral concentration, Ct values are determined as described in the Materials and Methods, and a Ct of zero indicates that the fluorescence signal did not cross the threshold limit within 45 cycles. n = 3 (A and B); n = 36 (C and D).

Figure 3

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection of novel coronarvirus N1 (nCoVN1) primer/probe target (A) and novel coronavirus N2 (nCoV-N2) primer/probe target (B). The x axis shows input RNA copies per well, and the y axis shows positive results across all parallel reactions performed. Diamonds are experimental data points resulting from replicate testing at given concentrations (x axis) (20 replicate reactions per datum point for RNA concentrations up to 1.0 copies per well and 3 replicates for RNA concentrations >1.0 copies per well). The inner bold line represents a Probit curve, with outer dotted lines representing 95% CIs. C: Limit of detection (LOD) calculated from the Probit analysis.