Sensitive Detection and Quantification of SARS-CoV-2 by Multiplex Droplet Digital RT-PCR

Abstract

Purpose. We aimed to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive detection of SARS-CoV-2 RNA with respect to human derived RNA and could be used for triage and monitoring of Covid-19 patients. Methods. A one step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19 negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. Results. Assay sensitivity of the RT-PCR assay drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared to water, while the sensitivity of the RT-ddPCR was not affected by the total NA background. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. Conclusion. The present study describes a robust and sensitive one-step RT-ddPCR multiplex assay for reliable detection and quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.

Cerebrospinal Fluid Analysis Post–COVID‐19 Is Not Suggestive of Persistent Central Nervous System Infection

“…13 Assays were carried out on LightCycler 480 (Roche Diagnostics, Mannheim, Germany). Samples with a weak signal in the RT-PCR assay were reanalyzed using a 1-step RT droplet digital (dd) PCR multiplex assay targeting SARS-CoV-2 E, RdRp, and N with a limit of detection of 5 viral RNA copies per reaction as previously described, 14 and 2 additional commercial tests. The Xpert Xpress SARS-CoV-2 (Cepheid, Sunnyvale, CA) with a limit of detection of 0.005 PFU/ml for N gene and 0.02 PFU/ml for E gene (PFU is defined as plaqueforming unit), and the Cobas SARS-CoV-2 assay on the automated Cobas 6800 (Roche Diagnostics) with a limit of detection of 0.0063 50% tissue culture infectious dose (TCID50)/ml for SARS-CoV-2 ORF1a/b and 0.0082 TCID50/ml for E gene were used.…”

Comparison of reverse-transcription qPCR and droplet digital PCR for the detection of SARS-CoV-2 in clinical specimens of hospitalized patients

“…The results of RT-qPCR and ddPCR performed using specimens collected from hospitalized COVID-19 patients demonstrate that ddPCR is indeed more sensitive than RT-qPCR. Moreover, negative specimens might be the result of poor sampling techniques, as the viral load in these patients varied significantly [ 5 , 6 , 8 , 13 , 15 ].…”

Identification and Validation of Common Reference Genes for Normalization of Esophageal Squamous Cell Carcinoma Gene Expression Profiles

“…Firstly, find eight commonly used reference genes in tumor tissue as candidate genes in the PubMed database. In addition to ACTB and GAPDH prevalently mentioned, there are ribosomal protein S18 (RPS18) [31,32], beta-2-microglobulin (B2M) [33,34], hypoxanthine phosphoribosyl transferase 1 (HPRT1) [35,36], glucuronidase beta (GUSB) [37][38][39][40], peptidylprolyl isomerase A (PPIA) [41,42], and phosphoglycerate kinase 1 (PGK1) [43,44]. Secondly, design and specificity analysis experiments were carried out to verify primer, qRT-PCR was performed, and the experimental results were evaluated by five kinds of expression stability software, such as GeNorm, NormFinder, and so on.…”