A RT-PCR assay for the detection of coronaviruses from four genera

Abstract

Background: During the past two decades, three novel coronaviruses (CoVs) have emerged to cause international human epidemics with severe morbidity. CoVs have also emerged to cause severe epidemics in animals. A better understanding of the natural hosts and genetic diversity of CoVs are needed to help mitigate these threats.

Objective: To design and evaluate a molecular diagnostic tool for detection and identification of all currently recognized and potentially future emergent CoVs from the Orthocoronavirinae subfamily.

Study design and results: We designed a semi-nested, reverse transcription RT-PCR assay based upon 38 published genome sequences of human and animal CoVs. We evaluated this assay with 14 human and animal CoVs and 11 other non-CoV respiratory viruses. Through sequencing the assay's target amplicon, the assay correctly identified each of the CoVs; no cross-reactivity with 11 common respiratory viruses was observed. The limits of detection ranged from 4 to 4 × 10² copies/reaction, depending on the CoV species tested. To assess the assay's clinical performance, we tested a large panel of previously studied specimens: 192 human respiratory specimens from pneumonia patients, 5 clinical specimens from COVID-19 patients, 81 poultry oral secretion specimens, 109 pig slurry specimens, and 31 aerosol samples from a live bird market. The amplicons of all RT-PCR-positive samples were confirmed by Sanger sequencing. Our assay performed well with all tested specimens across all sample types.

Conclusions: This assay can be used for detection and identification of all previously recognized CoVs, including SARS-CoV-2, and potentially any emergent CoVs in the Orthocoronavirinae subfamily.

Keywords: COVID-19; Coronavirus; Emerging; Infectious diseases; SARS-CoV-2.

Fig. 1

The phylogenetic tree of 38 coronaviruses with nucleotide sequences of RNA-dependent RNA polymerase, constructed by the neighbor-joining method using MEGA 7. The four coronavirus genera are grouped in shades of yellow (Alphacoronavirus), lightblue (Betacoronavirus), pink (Gammacoronavirus), and cyan (Deltacoronavirus). Seven human coronaviruses are marked in red.

Fig. 2

Multiple alignment of the of 38 available RdRp gene segments of coronaviruses, with nucleotide sequences and positions for the primers of the pan-CoV assay.

Fig. 3

Specificity analysis of pan-CoV assay using a panel of respiratory viral species. (A) Results of first amplification round (reverse transcription PCR). (B) Results of the conventional PCR for second step. Swabs 1 and 2 were collected from healthy people.

Fig. 4

Sensitivity tests of pan-CoV assay using serial dilutions of RNA and plasmid containing RdRp sequences of coronaviruses. (A) Sensitivity of pan-CoV assay using SARS-CoV plasmid. (B) Sensitivity of pan-CoV assay using mers-CoV plasmid. (C) Sensitivity of pan-CoV assay using SARS-CoV-2 RNA. The left and right sides of the DNA ladder are the results of first and second step of pan-CoV assay, respectively.