SARS-CoV-2 Diagnostics Based on Nucleic Acids Amplification: From Fundamental Concepts to Applications and Beyond

Abstract

COVID-19 pandemic ignited the development of countless molecular methods for the diagnosis of SARS-CoV-2 based either on nucleic acid, or protein analysis, with the first establishing as the most used for routine diagnosis. The methods trusted for day to day analysis of nucleic acids rely on amplification, in order to enable specific SARS-CoV-2 RNA detection. This review aims to compile the state-of-the-art in the field of nucleic acid amplification tests (NAATs) used for SARS-CoV-2 detection, either at the clinic level, or at the Point-Of-Care (POC), thus focusing on isothermal and non-isothermal amplification-based diagnostics, while looking carefully at the concerning virology aspects, steps and instruments a test can involve. Following a theme contextualization in introduction, topics about fundamental knowledge on underlying virology aspects, collection and processing of clinical samples pave the way for a detailed assessment of the amplification and detection technologies. In order to address such themes, nucleic acid amplification methods, the different types of molecular reactions used for DNA detection, as well as the instruments requested for executing such routes of analysis are discussed in the subsequent sections. The benchmark of paradigmatic commercial tests further contributes toward discussion, building on technical aspects addressed in the previous sections and other additional information supplied in that part. The last lines are reserved for looking ahead to the future of NAATs and its importance in tackling this pandemic and other identical upcoming challenges.

A Molecular Lateral Flow Assay for SARS-CoV-2 Quantitative Detection

“…However, the integration of nucleic acid amplification strategies such as RT-PCR, isothermal amplification, or CRISPR to LFAs [ 5 , 8 , 9 ] is necessary to enhance sensitivity, specificity, and detectability, as low diagnostic accuracy is the major hurdle of most of the commercially available point-of-care rapid tests [ 7 , 40 ]. In addition, compared with the conventional RT-PCR techniques and most LFA platforms with integrated amplification technologies, no RNA isolation from nasopharyngeal samples is needed, and a small volume of the sample in the supplied extraction buffer of a commercially available rapid antigen test is directly subjected to reverse transcription reaction that makes the procedure simpler [ 6 , 9 ]. However, amplification-free LFA platforms have been previously reported, as already mentioned, with high performance, but despite their obvious advantages related to the lack of RNA extraction, RT, and amplification steps, they show some limitations regarding, for example, their compromise of signal amplification potential by the limited binding sites and steric hindrance of the antibodies used [ 33 , 34 ].…”

“…and different analytical methodologies including RT-PCR, ELISA, lateral flow immunoassays (LFIA), chemiluminescence-based immunoassay (CLIA), loop-mediated isothermal amplification (LAMP), electrochemical assays, CRISPR-mediated technology, etc. [ 4 , 5 , 6 ]. Among these methodologies, RT-PCR and LFIA are currently used in clinical practice, with advantages and limitations related to sensitivity and specificity, cost, need for trained personnel and advanced instrumentation, assay time, portability and capability to be used for point-of-care testing, and patient self-administration [ 4 , 7 ].…”

Novel Lateral Flow-Based Assay for Simple and Visual Detection of SARS-CoV-2 Mutations

“…The COVID-19 outbreak has sparked the development of alternative NAAT strategies for fast, accurate, and affordable SARS-CoV-2 diagnostic tests ( Vindeirinho et al., 2022 ). Among these methods, nucleic acid lateral flow immunoassays (NALFIAs) based on isothermal amplification employing a lateral flow readout have been widely developed in a point-of-care format ( Zhang et al., 2022 ).…”

Reverse transcriptase-free detection of viral RNA using Hemo Klentaq DNA polymerase

“…A lot of severe diseases are caused by RNA viruses, such as influenza, AIDS, hepatitis A, C, D, E, and G, measles, poliomyelitis, Ebola haemorrhagic fever, etc. Recently, SARS-CoV-2 coronavirus has become a challenge caused the global COVID-19 pandemic and highlighted the need for the development of faster and more sensitive methods for detection of viral pathogens [ 2 , 3 ]. The demand for detection of SARS-CoV-2 RNA led to rapid elaboration of new advanced approaches based on isothermal amplification, microfluidics, CRISPR, biosensors, and other analytical techniques [ [4] , [5] , [6] , [7] , [8] , [9] , [10] ].…”

“…The demand for detection of SARS-CoV-2 RNA led to rapid elaboration of new advanced approaches based on isothermal amplification, microfluidics, CRISPR, biosensors, and other analytical techniques [ [4] , [5] , [6] , [7] , [8] , [9] , [10] ]. Nevertheless, polymerase chain reaction (PCR) still remains the gold standard and the most commonly used method for molecular diagnostics of infectious diseases [ 3 , 11 ].…”