Nucleic Acid Testing of SARS-CoV-2

Abstract

The coronavirus disease 2019 (COVID-19) has caused a large global outbreak. It is accordingly important to develop accurate and rapid diagnostic methods. The polymerase chain reaction (PCR)-based method including reverse transcription-polymerase chain reaction (RT-PCR) is the most widely used assay for the detection of SARS-CoV-2 RNA. Along with the RT-PCR method, digital PCR has emerged as a powerful tool to quantify nucleic acid of the virus with high accuracy and sensitivity. Non-PCR based techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) are considered to be rapid and simple nucleic acid detection methods and were reviewed in this paper. Non-conventional molecular diagnostic methods including next-generation sequencing (NGS), CRISPR-based assays and nanotechnology are improving the accuracy and sensitivity of COVID-19 diagnosis. In this review, we also focus on standardization of SARS-CoV-2 nucleic acid testing and the activity of the National Metrology Institutes (NMIs) and highlight resources such as reference materials (RM) that provide the values of specified properties. Finally, we summarize the useful resources for convenient COVID-19 molecular diagnostics.

The Production of Standardized Samples with Known Concentrations for Severe Acute Respiratory Syndrome Coronavirus 2 RT-qPCR Testing Validation for Developing Countries in the Period of the Pandemic Era

“…Of note, the sensitivity of the droplet digital PCR was found to be equal to or greater than that of the RT-qPCR [ 28 , 29 ]. These approaches, however, are not equivalent to RT-qPCR tests in terms of cost, sensitivity, or specificity [ 30 ]. Until now, the gold standard technique is the molecular diagnosis of SARS-CoV-2 using the RT-qPCR assay [ 13 , 31 ].…”

“…RT-qPCR assays necessitate the use of relatively expensive instruments as well as highly trained personnel. Thus, these provisions limit diagnostic capacity expansion in several countries [ 30 ]. Taken together, the validation of rapid diagnostic tests for COVID-19 should be a priority for diagnosis and follow-up of patients both in the hospital and in the community, allowing us to detect cases early and isolate patients and close contacts rapidly.…”

Diagnostic techniques for COVID-19: A mini-review

“… Broughton et al (2020) , De Smet et al (2021) , Fozouni et al (2020) , Han et al (2021) , Yoo et al (2021) and Zhang et al (2020a) .…”

Paper-based netlike rolling circle amplification (NRCA) for ultrasensitive and visual detection of SARS-CoV-2

“…In recent years, isothermal nucleic acid amplification technology has been widely used for rapid detection of virus-specific genes and other nucleic acid molecules [12] , [13] , [14] , [15] , [16] . At present, loop-mediated isothermal amplification (LAMP) [17] , [18] , DNA strand displacement amplification (SDA), clustered regularly interspaced short palindromic repeats (CRISPR)-mediated isothermal amplification technology [19] and nick endonuclease amplification reaction (NEAR) [20] have been successfully used in SARS-CoV-2 RNA detection [21] . Compared with other isothermal amplification techniques, NEAR is more convenient in primer design and has a high-speed reaction [22] .…”