Current Trends in RNA Virus Detection via Nucleic Acid Isothermal Amplification-Based Platforms

Abstract

Ribonucleic acid (RNA) viruses are one of the major classes of pathogens that cause human diseases. The conventional method to detect RNA viruses is real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), but it has some limitations. It is expensive and time-consuming, with infrastructure and trained personnel requirements. Its high throughput requires sophisticated automation and large-scale infrastructure. Isothermal amplification methods have been explored as an alternative to address these challenges. These methods are rapid, user-friendly, low-cost, can be performed in less specialized settings, and are highly accurate for detecting RNA viruses. Microfluidic technology provides an ideal platform for performing virus diagnostic tests, including sample preparation, immunoassays, and nucleic acid-based assays. Among these techniques, nucleic acid isothermal amplification methods have been widely integrated with microfluidic platforms for RNA virus detection owing to their simplicity, sensitivity, selectivity, and short analysis time. This review summarizes some common isothermal amplification methods for RNA viruses. It also describes commercialized devices and kits that use isothermal amplification techniques for SARS-CoV-2 detection. Furthermore, the most recent applications of isothermal amplification-based microfluidic platforms for RNA virus detection are discussed in this article.

Keywords: RNA virus detection; loop-mediated isothermal amplification (LAMP); nucleic acid sequence-based amplification (NASBA); point-of-care testing (POCT); recombinase polymerase amplification (RPA); recombinase-aided amplification (RAA).

Figure 1

Schematic illustrating saliva-based SARS-CoV-2 self-testing using RT-LAMP on a mobile device. Reprinted with permission from ref. [43]. Copyright (2022) American Chemical Society.

Figure 2

Schematic illustrating (a) the procedure of the DAMPR assay for SARS-CoV-2 detection; (b) principle of the DAMPR assay including RT-LAMP, G-quadruplex DNAzyme, and CRISPR-Cas9 reactions. Reprinted with permission from [33]. Copyright (2022) American Chemical Society.

Figure 3

Workflow of RT-LAMP-based electrochemical sensor in wastewater samples: (a) sampling from wastewater treatment plant; (b) nucleic acid extraction and concentration; (c) RT-LAMP mixtures for genetic amplification; (d) electrochemical monitoring of the RT-LAMP products via redox current. Reprinted with permission from ref. [44].

Figure 4

(a) Schematic illustration of the flow control of the centrifugal microfluidic device. (1) The device started with the dried RT-RPA reagents, primers, and probes. (2) A red dye buffer mixture and blue dye sample mixture were injected to their respective chambers. (3) The buffer mixture and the sample mixture burst into the mixing chamber, and then were mixed in both counterclockwise and clockwise directions. (4) The siphon finished priming via capillary reaction. (5) The mixture was transferred into the aliquoting chamber. (6) The mixture burst from the aliquoting chamber into the reaction chamber. (b) Sensitivity test results of an on-chip RT-RPA for the E, N, and ORF1ab genes of SARS-CoV-2 [62].

Figure 5

Illustration of the integrated microdroplet array platform for ultrafast and high-throughput diagnosis of SARS-CoV-2: (a) design and fabrication of the platform; (b) schematic of the RPA react reaction-based flow; (c) increase in the interaction between RPA components using ultrasound-driven microstreaming, enhancing the process of RPA. Reprinted with permission from ref. [65]. Copyright (2022) American Chemical Society.

Figure 6

Schematic diagram of the RPA reaction product detection using a lateral flow dipstick. (a) RPA-based generation of a specific amplicon for detection using a lateral flow (LF) assay. (b) Read-out of the RPA via LF. Reprinted and modified with permission from ref. [77].

Figure 7

Principle of norovirus digital nucleic acid detection. (a) Five key steps of method establishment: sequence collection, sequence alignment and analysis, design of primers and probe, RAA out of chip, and RAA on chip for the quantification process. (b) Schematic representation of the RAA probes followed by the generation of positive fluorescence signals. Reprinted and modified with permission from ref. [90].