The loop-mediated isothermal amplification method, coupled with reverse transcription (RT-LAMP), is the most widely used isothermal amplification technique for SARS-CoV-2 nucleic acid detection. First described by Notomi et al. [138], this method uses strand displacement activity of DNA polymerase and a set of inner and outer primers (four or six specific primer sequences) to amplify the target nucleic acids. LAMP is carried out at a single temperature between 60 and 65 °C, and generates up to 10⁹ copies of DNA in less than an hour ^{[133][137][139]}[133,137,139]. The LAMP procedure is initiated by hybridization of the forward inner primer (FIP) toward the target DNA template, which synthesizes the complementary strand. Then, the outer primer hybridizes to the target DNA, which initiates DNA synthesis by strand displacement. Subsequently, a FIP-hybridized complementary strand is released and forms a loop structure at one end of the sequence. The corresponding sequence becomes the template for the backward inner primer (BIP), which initiates another DNA synthesis by strand displacement, and then produces a ‘dumb-bell’ like DNA structure. Self-primed DNA synthesis of the corresponding sequence then converts the ‘dumb-bell’ like structure into a ‘stem-loop’ like DNA structure. Corresponding stem-loop DNA then becomes the template for LAMP cycling, and the target DNA sequence exponentially amplifies until the reaction is completed [138]. Amplified products are detected by changes of color as the accumulation of DNA changes, pH levels, or by changes in turbidity as magnesium pyrophosphate level increases ^{[140][141][142]}[140,141,142]. Amplified products are also detected by Calcein fluorescent dye or fluorescent intercalating dye ^[129][139][129,139]. The schematic procedure of RT-LAMP is shown as Figure 2.

Figure 2. Schematic procedure of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA). FIP = Forward Inner Primer, FOP = Forward Outer Primer, BIP = Backward Inner Primer, BOP = Backward Outer Primer.

Researchers have made efforts to optimize RT-LAMP for the development of rapid and sensitive detection of SARS-CoV-2. Several studies have evaluated the experimental parameters for RT-LAMP such as incubation temperature, incubation time, LoD, target genes, and primer sequences ^{[143][144][145]}[143,144,145]. Aside from optimizing the experimental parameters, researchers have developed modified RT-LAMP procedures including methods without prior RNA extraction steps and high-throughput colorimetric assay methods using a 96-well plate format ^[146][147][146,147]. Modified RT-LAMP procedures also include methods coupled with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, a nanoparticle-based biosensor, and artificial intelligence ^{[148][149][150][151]}[148,149,150,151].

3.2. Recombinase Polymerase Amplification (RPA)

Recombinase polymerase amplification is another isothermal amplification method that is widely used for SARS-CoV-2 detection. First described by Piepenburg et al. [152], RPA uses a complex of recombinase and two target specific primers (forward and reverse primers) to amplify the target nucleic acids [152]. Once the target nucleic acids are identified, recombinase-primer complex unwinds the target DNA and allows forward and reverse primers to hybridize [153]. The displaced DNA strand is amplified in the presence of DNA polymerase as primers elongate, and the template DNA is exponentially amplified until the reaction is completed [153]. RPA reaction is carried out at a single temperature between 37 and 42 °C, and the reaction is completed when ATPs are depleted, typically in less than an hour [154]. Amplified products are detected by gel electrophoresis, antigenic tags on primers and tag-specific antibodies, or fluorescent signals produced by a conjugated fluorophore and quencher on primers ^{[152][153][154]}[152,153,154]. The schematic procedure of reverse transcription RPA (RT-RPA) is shown as Figure 2.

For SARS-CoV-2 detection, researchers have optimized RPA procedures by testing various experimental parameters that can now detect less than five viral copies from patient samples within 45 min from the sample collection [134]. RPA methods have also been optimized for SARS-CoV-2 detection by coupling RPA-based amplification with various CRISPR-based detection methods ^{[155][156][157]}[155,156,157].

3.3. Other Isothermal Nuleic Acid Amplification Methods

Other than LAMP and RPA, isothermal amplification methods such as NASBA, SDA, and RCA have been used for the detection of SARS-CoV-2 ^{[136][137][158]}[136,137,158]. Although we will not describe each technique in detail here, Table 3 presents the general features and components of each isothermal amplification method.

Table 3. General features of the isothermal amplification techniques for SARS-CoV-2 detection.

Method	Components	Temperature	Time	Detection Method	Advantages *	Disadvantages *
Loop-mediated isothermal amplification (LAMP)	DNA polymerase, forward inner primer, backward inner primer, forward outer primer, backward outer primer	60–65 °C	>1 h	Colorimetric, turbimetric, fluorescence probe, intercalating dye	High specificity. Less sensitive to inhibitors in biological samples	False positive in negative control
Recombinase polymerase amplification (RPA)	Recombinase, single stranded binding protein, DNA polymerase, forward primer, reverse primer	37–42 °C	>1 h	Fluorescence, antigenic-tag (antibody)	Performed in the presence of PCR inhibitors. Fast and sensitive	Inhibited by detergents (SDS and CTAB). Non-specific/high background signal
Nucleic acid sequence-based amplification (NASBA)	RNase H, reverse transcriptase, T7 DNA-dependent RNA polymerase, forward primer with T7 promoter sequence, reverse primer	41 °C	>2 h	Fluorescence	More sensitive and less time-consuming	Non-specific reactions/false positives
Strand-displacement amplification (SDA)	DNA polymerase, restriction endonuclease, primers, dCTP, dTTP, dGTP, dATPα	37–49 °C	>2 h	Fluorescence	High specificity. Detection of large RNA molecules	Non-specific reaction/high background signal
Rolling circle amplification (RCA)	DNA ligase, DNA polymerase, primer, padlock probe	30–37 °C	>1.5 h	Fluorescence	High specificity	False negatives and false positives

* Advantages and disadvantages in comparison with RT-qPCR methods.