A Novel Multiplex qRT-PCR Assay to Detect SARS-CoV-2 Infection: High Sensitivity and Increased Testing Capacity.

Sara Petrillo,Miriam Martini,Alessandro Giorgio,Jean Piero Margaria,Davide Barberio,Giovanna Carrà,Maria Chiara De Santis,Rossana Cavallo,Paolo Bottino,Elisa Zanotto,Fiorella Altruda,Giorgia Mandili

doi:10.3390/microorganisms8071064

Abstract

Rapid and sensitive screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to limit the spread of the global pandemic we are facing. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is currently used for the clinical diagnosis of SARS-CoV-2 infection using nasopharyngeal swabs, tracheal aspirates, or bronchoalveolar lavage (BAL) samples. Despite the high sensitivity of the qRT-PCR method, false negative outcomes might occur, especially in patients with a low viral load. Here, we developed a multiplex qRT-PCR methodology for the simultaneous detection of SARS-CoV-2 genome (N gene) and of the human RNAse P gene as internal control. We found that multiplex qRT-PCR was effective in detecting SARS-Cov-2 infection in human specimens with 100% sensitivity. Notably, patients with few copies of SARS-CoV-2 RNA (<5 copies/reaction) were successfully detected by the novel multiplex qRT-PCR method. Finally, we assessed the efficacy of multiplex qRT-PCR on human nasopharyngeal swabs without RNA extraction. Collectively, our results provide evidence of a novel and reliable tool for SARS-CoV-2 RNA detection in human specimens, which allows the testing capacity to be expanded and the RNA extraction step to be bypassed.

Highlights

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus, was recently identified in patients with acute respiratory disease and declared by the World Health Organization (WHO) in March 2020 as global pandemic [1]
We found that multiplex Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was effective in detecting SARS-CoV-2 infection, to singleplex qRT-PCR assays performed on the same target sequences
The human RNase P gene was included as internal control to evaluate the quality of clinical specimens and nucleic acid extraction

Summary

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus, was recently identified in patients with acute respiratory disease and declared by the World Health Organization (WHO) in March 2020 as global pandemic [1] (https://www.who.int/emergencies/diseases/novelcoronavirus-2019). Several problems have emerged in clinic diagnostics, in scaling up daily tests and managing the lack of supplies for extraction and evaluation reagents [2]. This situation promoted the search for alternative protocols to ensure continuity in samples screening. The Centers for Disease Control and Prevention (CDC) in the United States rapidly developed a qRT-PCR protocol to detect SARS-CoV-2 infection in patients. This protocol is based on separate qRT-PCR reactions targeting the SARS-CoV-2-specific N gene [4]. We investigated the detection efficacy of the multiplex qRT-PCR assay on swab transport medium (termed diluent hereafter) without RNA extraction

Methods

Results

Discussion

Conclusion