Multiplexed Magnetofluorescent Bioplatform for the Sensitive Detection of SARS-CoV-2 Viral RNA without Nucleic Acid Amplification

Abstract

Rapid and sensitive detection of SARS-CoV-2 virus genetic material is of paramount importance to mitigate the COVID-19 pandemic outbreak and lower the death toll. Herein, we report the design of a magnetofluorescent bioplatform for the direct and specific detection of the viral RNA of SARS-CoV-2 in the total RNA extracted from nasopharyngeal swabs of COVID-19-positive patients. A higher fluorescence response was achieved using two capture probes tethered to magnetic beads using a biotin/streptavidin linkage, targeting two specific sites in the ORF1a and S genes. Two horseradish peroxidase (HRP)-conjugated reporter sequences, complementary to the loci of the S and N genes, were used to reveal the presence of the viral RNA through the oxidation of o -phenylenediamine to fluorescent 2,3-diaminophenazine. Under optimal conditions, the bioplatform showed high selectivity and sensitivity and was able to detect as low as 0.01 ng of viral RNA (1 × 10 3 copies/μL) with a linear dynamic range varying from 0.01 to 3.0 ng (1 × 10 3 to 9 × 10 7 copies/μL). The bioplatform was also able to discriminate the SARS-CoV-2 RNA from those of other related viruses such as hepatitis C, West Nile, measles, and non-polio viruses. Furthermore, the developed biosensor was validated in 46 clinical samples (36 COVID-19-positive patients and 10 COVID-19-negative subjects, as assessed with the gold standard RT-qPCR method). Both sensitivity and specificity of the developed method reached 100%. Finally, making such a simple and specific method available in the field, at a primary point of care, can better help the detection of SARS-CoV-2 infection in low-resource settings.

A Study of the Detection of SARS-CoV-2 ORF1ab Gene by the Use of Electrochemiluminescent Biosensor Based on Dual-Probe Hybridization

“…The method can meet the requirements for analyzing simulated samples such as saliva and urine, with the advantages of high sensitivity, stable reproducibility, and anti-interference solid abilities. Compared with the existing reported detection method for the SARS-CoV-2 gene by ECL biosensor based on the amplification technique [ 24 , 25 ] and other biosensors [ 26 , 27 , 28 , 29 , 30 , 31 ], the ECL biosensor based on dual-probe hybridization detection of SARS-CoV-2 has improved sensitivity, as shown in Table 4 . It also has the advantages of simplifying the operation steps without amplifying and shortening the detection time, achieving rapid and highly sensitive detection within 30 min.…”

Sandwich-Based Immunosensor for Dual-Mode Detection of Pathogenic F17–Positive Escherichia coli Strains

“…Biosensors have been one of the most attractive detection techniques due to their excellent performance, namely cost-effectiveness, fast response, high sensitivity, and high selectivity [ 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. Immunosensors are a very attractive tool for the detection of pathogenic bacteria.…”

Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

“…In addition to conventional nucleic acid amplification reactions that we described previously, alternative multiplex virus diagnostic testing assays have also been developed to directly identify the viral targets present to reduce the complexity, cost, and possible contamination [ 12 , 83 , 84 , 85 , 86 , 87 ]. For example, a magnetofluorescent bio-platform for direct detection of SARS-CoV-2 is demonstrated by Zayani et al Using a nasopharyngeal swab sample directly, the ORF1a and S gene probes are tethered to magnetic beads with a biotin/streptavidin linkage to extract the RNA target.…”