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Quantitative and ultrasensitive in situ immunoassay technology for SARS-CoV-2 detection in saliva.

ABSTRACT: The coronavirus disease 2019 (COVID-19) pandemic has become an immense global health crisis. However, the lack of efficient and sensitive on-site testing methods limits early detection for timely isolation and intervention. Here, we present a quantitative and ultrasensitive in situ immunoassay technology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in saliva (QUIT SARS-CoV-2). Our nanoporous membrane resonator generates a rapid oscillating flow to purify and concentrate fully intact SARS-CoV-2 virus in saliva by 40-fold for in situ detection of viral antigens based on chemiluminescent immunoassay within 20 min. This method can not only achieve a detection sensitivity below 100 copies/ml of virus, comparable to the bench-top PCR equipment; it can also improve detection specificity via direct monitoring of viral loads. The integrated portable QUIT SARS-CoV-2 system, which enables rapid and accurate on-site viral screening with a high-throughput sample pooling strategy, can be performed in primary care settings and substantially improve the detection and prevention of COVID-19.

SUBMITTER: Chen Y

PROVIDER: S-EPMC9132547 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Quantitative and ultrasensitive in situ immunoassay technology for SARS-CoV-2 detection in saliva.

Chen Yuchao Y Liu Fei F Lee Luke P LP

Science advances 20220525 21

The coronavirus disease 2019 (COVID-19) pandemic has become an immense global health crisis. However, the lack of efficient and sensitive on-site testing methods limits early detection for timely isolation and intervention. Here, we present a quantitative and ultrasensitive in situ immunoassay technology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in saliva (QUIT SARS-CoV-2). Our nanoporous membrane resonator generates a rapid oscillating flow to purify and concent ...[more]

PMID: 35613342

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Project description:A large-scale diagnosis of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is essential to downregulate its spread within as well as across communities and mitigate the current outbreak of the pandemic novel coronavirus disease 2019 (COVID-19). Herein, we report the development of a rapid (less than 5 min), low-cost, easy-to-implement, and quantitative paper-based electrochemical sensor chip to enable the digital detection of SARS-CoV-2 genetic material. The biosensor uses gold nanoparticles (AuNPs), capped with highly specific antisense oligonucleotides (ssDNA) targeting viral nucleocapsid phosphoprotein (N-gene). The sensing probes are immobilized on a paper-based electrochemical platform to yield a nucleic-acid-testing device with a readout that can be recorded with a simple hand-held reader. The biosensor chip has been tested using samples collected from Vero cells infected with SARS-CoV-2 virus and clinical samples. The sensor provides a significant improvement in output signal only in the presence of its target-SARS-CoV-2 RNA-within less than 5 min of incubation time, with a sensitivity of 231 (copies ?L-1)-1 and limit of detection of 6.9 copies/?L without the need for any further amplification. The sensor chip performance has been tested using clinical samples from 22 COVID-19 positive patients and 26 healthy asymptomatic subjects confirmed using the FDA-approved RT-PCR COVID-19 diagnostic kit. The sensor successfully distinguishes the positive COVID-19 samples from the negative ones with almost 100% accuracy, sensitivity, and specificity and exhibits an insignificant change in output signal for the samples lacking a SARS-CoV-2 viral target segment (e.g., SARS-CoV, MERS-CoV, or negative COVID-19 samples collected from healthy subjects). The feasibility of the sensor even during the genomic mutation of the virus is also ensured from the design of the ssDNA-conjugated AuNPs that simultaneously target two separate regions of the same SARS-CoV-2 N-gene.

Project description:BackgroundA previous study demonstrated the performance of the Salivette® (SARSTEDT, Numbrecht, Germany) as a homogeneous saliva collection system to diagnose COVID-19 by RT-qPCR, notably for symptomatic and asymptomatic patients. However, for convalescent patients, the corroboration of molecular detection of SARS-CoV-2 in paired nasopharyngeal swabs (NPS) and saliva samples was unsatisfactory.ObjectivesThe aim of the present work was to assess the concordance level of SARS-CoV-2 detection between paired sampling of NPSs and saliva collected with Salivette® at two time points, with ten days of interval.ResultsA total of 319 paired samples from 145 outpatients (OP) and 51 healthcare workers (HW) were collected. Unfortunately, at day ten, 73 individuals were lost to follow-up, explaining some kinetic missing data. Due to significant waiting rates at hospitals, most of the patients ate and/or drank while waiting for their turn. Consequently, mouth washing was systematically proposed prior to saliva collection. None of the HW were diagnosed as SARS-CoV-2 positive using NPS or saliva specimens at both time points (n = 95) by RT-qPCR. The virus was detected in 56.3% (n = 126/224) of the NPS samples from OP, but solely 26.8% (n = 60/224) of the paired saliva specimens. The detection of the internal cellular control, the human RNase P, in more than 98% of the saliva samples, underlined that the low sensitivity of saliva specimens (45.2%) for SARS-CoV-2 detection was not attributed to an improper saliva sample storing or RNA extraction.ConclusionsThis work revealed that mouth washing decreased viral load of buccal cavity conducting to impairment of SARS-CoV-2 detection. Viral loads in saliva neo-produced appeared insufficient for molecular detection of SARS-CoV-2. At the time when saliva tests could be a rapid, simple and non-invasive strategy to assess large scale schoolchildren in France, the determination of the performance of saliva collection becomes imperative to standardize procedures.

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Quantitative and ultrasensitive in situ immunoassay technology for SARS-CoV-2 detection in saliva.

Publications

Quantitative and ultrasensitive in situ immunoassay technology for SARS-CoV-2 detection in saliva.

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