Abstract
BackgroundThe ongoing outbreak of the novel human coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (also known as 2019-nCoV) has become a global health concern. Rapid and easy-to-use diagnostic techniques are urgently needed to diagnose SARS-CoV-2 infection.MethodsWe devised a reverse transcription multiple cross-displacement amplification (RT-MCDA) coupled with a nanoparticle-based biosensor assay (RT-MCDA-BS) for rapid, sensitive and specific diagnosis of coronavirus disease 2019 (COVID-19). Two primer sets were designed to target the open reading frame 1a/b and nucleoprotein gene of SARS-CoV-2. A total of 183 clinical samples, including 65 patients with COVID-19 infection and 118 patients with other pathogen infections were used to testify the assay's feasibility. Diagnosis results were reported visually using the biosensor.FindingsThe assay designed was performed using a simple instrument which could maintain the reaction in a constant temperature at 64°C for only 35 min. The total COVID-19 RT-MCDA-BS test procedure could be finished within 1 h. The COVID-19 RT-MCDA-BS could detect down to five copies of target sequences. Among 65 clinical samples from the COVID-19 patients, 22 (33.8%) positive results were obtained from faeces, nasal, pharyngeal and anal swabs via COVID-19 RT-MCDA-BS assay, while real-time reverse transcription-PCR assay only detected 20 (30.7%) positive results in these samples. No positive results were obtained from clinical samples with non-COVID-19 infections.InterpretationCOVID-19 RT-MCDA-BS was a rapid, reliable, low-cost and easy-to-use assay, which could provide an attractive laboratory tool to diagnose COVID-19 in multiple clinical specimens, especially for field, clinic laboratories and primary care facilities in resource-poor settings.
Highlights
In late December 2019, an outbreak of pneumonia caused by a novel human coronavirus (severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); referred to 2019-nCov) emerged in Wuhan, in the Hubei province in China [1, 2]
Our COVID-19 reverse transcription multiple cross-displacement amplification (RT-multiple cross-displacement amplification (MCDA))-BS assay detected 22 (33.8%) positive results from different types of clinical specimens, while real-time RT (rRT)-PCR only found 20 (30.7%) positive results
The findings suggest that our COVID-19 reverse transcription (RT)-MCDA-BS assay is more powerful in detecting COVID-19 patients, especially those with very low virus loads
Summary
In late December 2019, an outbreak of pneumonia caused by a novel human coronavirus (severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); referred to 2019-nCov) emerged in Wuhan, in the Hubei province in China [1, 2]. A rapid, easy-to-use and reliable test tool was urgently required for diagnosing infections, treating the patients and controlling the spreading of COVID-19. The definitive diagnosis of COVID-19 infections strongly relies on PCR-based methods, such as reverse transcription (RT)-PCR and real-time RT (rRT)-PCR [6]. They require expensive thermal cycle apparatus and very skilful laboratory workers. The 62% positive rate obtained by PCR-based technologies because of their sensitivity limitation among clinically diagnosed COVID-19 patients is not effective for infection control [6, 7]. Methods: We devised a reverse transcription multiple cross-displacement amplification (RT-MCDA) coupled with a nanoparticle-based biosensor assay (RT-MCDA-BS) for rapid, sensitive and specific diagnosis of coronavirus disease 2019 (COVID-19). Interpretation: COVID-19 RT-MCDA-BS was a rapid, reliable, low-cost and easy-to-use assay, which could provide an attractive laboratory tool to diagnose COVID-19 in multiple clinical specimens, especially for field, clinic laboratories and primary care facilities in resource-poor settings
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