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.

Fig. 1.. Illustration of the QUIT SARS-CoV-2 system and workflow.

(A) Illustration showing saliva collection for SARS-CoV-2 virus detection. (B) Illustration of the QUIT SARS-CoV-2 system including a workstation and two disposable purification and detection devices. (C) Illustration of the detection module in a QUIT SARS-CoV-2 system. AP, air pressure; NP, negative pressure; PMT, photomultiplier tube. (D) Illustration showing the workflow for virus purification, enrichment, and detection using the QUIT SARS-CoV-2 system. CL, chemiluminescence; HRP, horseradish peroxidase.

Fig. 2.. Validation and application of the QUIT SARS-CoV-2 system.

(A) Comparison of RT-qPCR Ct values from samples with and without enrichment by the QUIT SARS-CoV-2 system. SARS-CoV-2 viruses spiked into pooled saliva from healthy donors were used as study samples. (B) Characterization of QUIT SARS-CoV-2 using pooled saliva samples spiked with SARS-CoV-2 virus at different concentrations (red curve present as RLU values). RT-qPCR was carried out for comparison (black curve present as Ct values). Ct value was recorded as 45 when no signal was detected. Pooled saliva without spiked virus (0 copies/ml) was used as negative controls. (C) Chemiluminescent signals detected by a PMT at different viral loads. The curves were fitted by the sigmoid function. (D) Coefficient of variation (CV) for the signal intensities on the plateau collected in 30 s. (E) Comparison of the detection results for intact virus and lysed virus after enrichment by the QUIT SARS-CoV-2 system. Chemiluminescent detection by QUIT SARS-CoV-2 and RT-qPCR was used for the characterization of viral antigen and RNA. (F) Illustration showing the luminescent signals detected by QUIT SARS-CoV-2 including the detection of internal control and virus. AP, alkaline phosphatase. (G) Application of QUIT SARS-CoV-2 in the study of COVID-19 patient samples and healthy controls. Mann-Whitney test was used to calculate the P value. RLU, relative light unit.