One-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification.

Droplet digital polymerase chain reaction (ddPCR) is a third generation of PCR that was recently developed to overcome the limitation of direct quantification observed in real-time quantification PCR (qPCR). Recent studies have shown that ddPCR is more sensitive than the gold standard reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples. In combination with multiplexing, multiple RT-ddPCR assays can be developed to directly quantify different SARS-CoV-2 nucleic acid targets within a single sample, significantly saving on cost and time. Since ddPCR is tolerant to a number of inhibitors unlike qPCR, it can be used to detect and quantify samples from complex environments like wastewater. Here we present three one-step RT-ddPCR protocols on how to develop simplex (one target), duplex (two targets), and triplex probe mix (three targets) assays for SARS-CoV-2 detection and quantification. The assays can be used for diagnosis or other research-related SARS-CoV-2 applications.

Digital PCR Applications in the SARS-CoV-2/COVID-19 Era: a Roadmap for Future Outbreaks.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global public health disaster. The current gold standard for the diagnosis of infected patients is real-time reverse transcription-quantitative PCR (RT-qPCR). As effective as this method may be, it is subject to false-negative and -positive results, affecting its precision, especially for the detection of low viral loads in samples. In contrast, digital PCR (dPCR), the third generation of PCR, has been shown to be more effective than the gold standard, RT-qPCR, in detecting low viral loads in samples. In this review article, we selected publications to show the broad-spectrum applications of dPCR, including the development of assays and reference standards, environmental monitoring, mutation detection, and clinical diagnosis of SARS-CoV-2, while comparing it analytically to the gold standard, RT-qPCR. In summary, it is evident that the specificity, sensitivity, reproducibility, and detection limits of RT-dPCR are generally unaffected by common factors that may affect RT-qPCR. As this is the first time that dPCR is being tested in an outbreak of such a magnitude, knowledge of its applications will help chart a course for future diagnosis and monitoring of infectious disease outbreaks.

SARS-CoV-2/COVID-19 Laboratory Biosafety Practices and Current Molecular Diagnostic Tools.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/coronavirus disease 2019 (COVID-19) pandemic has crippled several countries across the globe posing a serious global public health challenge. Despite the massive rollout of vaccines, molecular diagnosis remains the most important method for timely isolation, diagnosis, and control of COVID-19. Several molecular diagnostic tools have been developed since the beginning of the pandemic with some even gaining emergency use authorization (EUA) from the United States (US) Food and Drug Administration (FDA) for in vitro diagnosis of SARS-CoV-2. Herein, we discuss the working principles of some commonly used molecular diagnostic tools for SARS-CoV-2 including nucleic acid amplification tests (NAATs), isothermal amplification tests (IATs), and rapid diagnostic tests (RDTs). To ensure successful detection while minimizing the risk of cross-infection and misdiagnosis when using these diagnostic tools, laboratories should adhere to proper biosafety practices. Hence, we also present the common biosafety practices that may ensure the successful detection of SARS-CoV-2 from specimens while protecting laboratory workers and non-suspecting individuals from being infected. From this review article, it is clear that the SARS-CoV-2 pandemic has led to an increase in molecular diagnostic tools and the formation of new biosafety protocols that may be important for future and ongoing outbreaks.

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification.

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing.

Introduction: With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold-standard reverse transcription PCR (RT-PCR) in SARS-CoV-2 detection. However, few studies have been reported on developing multiplex ddPCR assays for SARS-CoV-2 detection and their performance. This study shows steps on how to develop different ddPCR SAR-CoV-2 assays including higher order multiplex assays for SARS-CoV-2 detection and antiviral screening.Methods: Using multiple primer/probe sets, we developed, optimized, and analyzed the performance of simplex (1 target), duplex (2 targets), triplex probe mix (3 targets), and quadruplex (4 targets) SARS-CoV-2 ddPCR assays based on a two-color ddPCR detection system.Results: Results showed that the quadruplex assay had similar limits of detection and accuracy to the lower multiplex assays. Analyzing 94 clinical samples demonstrated that the ddPCR triplex probe mix assay had better sensitivity than the RT-qPCR assay. Additionally, the ddPCR multiplex assay showed that remdesivir could inhibit the growth of SARS-CoV-2 in vitro while another testing drug could not.Conclusion: Our research shows that developing multiplex ddPCR assays is possible by combing probe mix and amplitude-based multiplexing, which will help in developing multiplexed ddPCR assays for different SARS-CoV-2 applications.