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

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.

Summary of the Detection Kits for SARS-CoV-2 Approved by the National Medical Products Administration of China and Their Application for Diagnosis of COVID-19

“…rRT-PCR has played an important role in early detection, and is considered the 'gold standard' for COVID-19 testing. However, false negative results due to inappropriate sample collection timing, sample type, sampling technique or other problems have limited its usage and thus increased the importance of serological testing (Coupeau et al 2020;Lee et al 2020;Petrillo et al 2020;Wolff et al 2020). Serological assays can detect previous SARS-CoV-2 infection and provide information on the progression of the disease, but the tests must be performed within the correct time frame after the onset of the disease (Arun Krishnan et al 2020;Carinci et al 2020;Lee et al 2020;.…”

“…After infecting a human, SARS-CoV-2 amplifies quickly. Therefore, nucleic acids of the virus can be detected from the early stage in samples such as nasopharynx swabs, oropharynx swabs, sputum, and stool Paoli et al 2020;Petrillo et al 2020;Yan et al 2020). Among all approved nucleic acid detection kits, the novel rRT-PCR techniques were developed in rapid response to the emergence of COVID-19 in China.…”

“…Therefore, nucleic acids of the virus can be detected from the early stage in samples such as nasopharynx swabs, oropharynx swabs, sputum, and stool (Li and Ren 2020 ; Paoli et al 2020 ; Petrillo et al . 2020 ; Yan et al . 2020 ).…”

Current methods and prospects of coronavirus detection

“…Real time reverse transcription polymerase chain reaction (RT-PCR), as a specific and simple quantitative detection method [ 67 , 68 ], has been widely used in the detection of various viruses [ [69] , [70] , [71] , [72] ]. For example, Nunes et al used RT-qPCR to detect four Brazilian Amazon hantaviruses with a detection limit of 0.9 copies/μL.…”

COVID-19 case management: The policy model in Morocco context

“…Each one who had no more symptoms needs two tests noticing negative results or close to the Cq >34 (do not have meaningful transmissibility of the disease) [36]. Indeed, Cq > 34 correspond to less than four viral copies in RT-qPCR device with 100% sensitivity [37,38]. A French team confirmed the strong correlation between successful isolation of SARS-CoV-2 in cell culture and Cq value of RT-qPCR targeting envelop gene and suggested that patients with Cq above 34 (Based on their PCR device used) are not contagious and can be exempted from hospital care or for strict confinement of the non-hospitalised patients [39].…”