Nucleic acid testing of SARS-CoV-2: A review of current methods, challenges, and prospects

Abstract

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has brought a huge threat to public health and the global economy. Rapid identification and isolation of SARS-CoV-2-infected individuals are regarded as one of the most effective measures to control the pandemic. Because of its high sensitivity and specificity, nucleic acid testing has become the major method of SARS-CoV-2 detection. A deep understanding of different diagnosis methods for COVID-19 could help researchers make an optimal choice in detecting COVID-19 at different symptom stages. In this review, we summarize and evaluate the latest developments in current nucleic acid detection methods for SARS-CoV-2. In particular, we discuss biosensors and CRISPR-based diagnostic systems and their characteristics and challenges. Furthermore, the emerging COVID-19 variants and their impact on SARS-CoV-2 diagnosis are systematically introduced and discussed. Considering the disease dynamics, we also recommend optional diagnostic tests for different symptom stages. From sample preparation to results readout, we conclude by pointing out the pain points and future directions of COVID-19 detection.

Bioluminescent Intercalating Dyes for Ratiometric Nucleic Acid Detection

“…Sensitive detection of nucleic acids is key to a wide array of applications, ranging from infectious disease diagnosis and oncology to food and environmental safety monitoring and forensic DNA fingerprinting. − Polymerase chain reaction (PCR)-based assays set the benchmark for nucleic acid detection, owing to their ability to amplify as few as 1–10 copies of target DNA to detectable concentrations . To facilitate quantification of target DNA, quantitative PCR (qPCR) allows for real-time detection utilizing fluorescent intercalating dyes that exhibit a strong increase in fluorescence upon binding nonspecifically to double-stranded DNA (dsDNA) .…”

Democratizing nucleic acid-based molecular diagnostic tests for infectious diseases at resource-limited settings – from point of care to extreme point of care

“…The nucleic acid amplification tests (NAAT), which analyse the RNA or DNA from appropriate body-fluid samples to detect the genetic sequences specific to the invading pathogen, represent the most preferred molecular diagnostic procedures from the scientific and technological robustness considerations, where the nucleic acid is amplified in terms of the number of its copies (just as multiplex photocopies in a photo-copying machine) as the test progresses, enabling the detection of the target gene sequence at some level of amplification that can resolve the specific region of interest of the concerned gene. 4,5…”

Cycle Threshold (Ct) Values of SARS-CoV-2 Detected with the GeneXpert® System and a Mutation Associated with Different Target Gene Failure

“…Nucleic acid detection tests are used to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and several devices and reagent kits are commercially available for this purpose [1][2][3][4]. Among these, real-time reverse transcription polymerase chain reaction (qRT-PCR) assays have formed the backbone of coronavirus disease-19 (COVID- 19) diagnosis since the start of the COVID-19 pandemic, and many qRT-PCR-based SARS-CoV-2 testing platforms have been developed and made commercially available, including the GeneXpert ® System and Cepheid Xpert Xpress SARS-CoV-2 assay kit (GX) (Beckman Coulter, Inc., Brea, CA, USA) [5], which target the envelope gene (E) and N2 region of the nucleocapsid gene (N2) of SARS-CoV-2.…”