*2.1.4 Multiplex SDA reaction*

The detection of more than one target from a single sample is usually required in medical diagnosis. Multiplexing is mainly achieved using spatial separation of targets, regional separation by targeting specific sites, or label-based techniques [53]. Though spatial and regional separation methods are highly employed for on-site multiplexed detection of various targets, they still suffer from expensive apparatus and complicated procedures [54, 55]. An alternative method is target-labeling approach, which uses different molecular recognition elements (dyes, enzymes, DNA probes, beads, aptamers, etc.) to identify different targets.-

Primer design and optimization are essential for multiplex SDA.-Singleplex SDA requires four- primers to detect one target; therefore, duplexed detection would require eight primers, and- so on. Thus, the number of primers increases rapidly with the number of targets. The large- number of primers increases the complexity of the multiplex amplification system and therefore- decreases the stability of the system. To solve this problem, Walker etal. developed a method- called "adapter-mediated duplex SDA" for simultaneous detection of *Mycobacterium species*  using fewer primers and without altering amplification yield [20]. In this method, a single pair- of amplification primers and adapter sequences is used, where two target strands are amplified- exponentially using dual primers. The first primer is attached to one end of the second target,- while the second primer is appended to one end of the first target sequence. After amplification- of the target strands by the primers, the adapter sequences start to bind to the amplified target- sequences and begin extension and displacement, which results in a cascade of exponential- amplification of the target sequences using the adapters rather than the primers. This method- was also used for multiplexed SDA of three distinct DNA sequences of *Mycobacterium tuberculosis* and other mycobacteria [56]. Furthermore, multiplex SDA was used to amplify multiple- SNPs simultaneously with molecular beacon probe-assisted fluorescent signal readout [57].- Most recently, several *BRCA* mutations were genotyped by combining SDA and mass spectrometry. Allele-specific regions were amplified and then ligated to adapters by DNA ligase,- and the ligated products were SDA amplified with universal primers. The resulting fragments- were analyzed and confirmed using mass spectrometry [58]. Though, this SDA method required- complex equipment, it was able to detect hundreds of mutations.-

### *2.1.5 Sample preparation*

Isothermal amplification usually requires specimen isolation and culture, enzymatic treatment or genomic DNA (gDNA) extraction, and/or heat denaturation (in case of dsDNA) to obtain template DNA.-Traditional sample preparation methods like genomic DNA extraction are time consuming and susceptible to contamination. Thus, crude cell lysate method is widely used, whereby cells are heat killed or enzymatically pretreated using proteinase K to expose DNA ready for amplification [59]. Cell cultures are usually heated at 92–95°C for 3–5min and cooled to appropriate SDA temperature prior to the reaction. An up to date approach termed HUDSON (heating unextracted diagnostic samples to obliterate nucleases) combines the dual range of heating, that is, one temperature (37–50°C, 5–20min) for nuclease inactivation, and another temperature (64–95°C, 5–20min) for pathogen inactivation and genome exposure [60]. Though heat-assisted amplification is still frequently preferred owing to its simplicity, but it cannot differentiate nucleic acids from live and dead cells. As solution, Tong and colleagues used a set of restriction enzymes and modified nucleotides that could target different sites of target template and create a site of nicking and extension for SDA target DNA amplification [61]. However, these modified nucleotides and probes are costly, and the restriction enzymes exhibit random target digestion, which results in high background, low sensitivity, and specificity. Alternatively, heat treatment can also be abolished by using an improved SDA approach that initiates amplification at a DNA breaching site (Hoogsteen pairing), though its specificity to long templates is still of concern [3, 62].-

To overcome contamination and detect live targets accurately, gDNA extraction free isothermal amplification methods have been adopted, which use extracellular compartment recognizing moleculessuch as aptamers. We reported ultrasensitive aptamer-based biosensors for the detection of live pathogens including *E. coli* and *S. enteritidis* [15, 24, 36]*.* In these methods, a dual aptamer system recognizing two extracellular membrane components are used. One bacterium-targeting aptamer is modified with biotin in order to react with streptavidincoated magnetic beads during positive selection, while another bacterium-targeting aptamer is used as a template for SDA amplification. The bacteria-aptamer-magnetic bead complex is enriched, amplified, and applied to lateral flow strip for visual detection (**Figure 2**). This method sensitivity to pathogens is 10*cfu* of live cells, therefore reduces false-positive results.- Strand Displacement Amplification for Multiplex Detection of Nucleic Acids 69 http://dx.doi.org/10.5772/intechopen.80687

**Figure 2.** Conceptual illustration of SDA and aptamer-based nucleic acid biosensor**.** Two target-specific aptamers termed biotinylated capture aptamers (C-aptamer) and amplifying aptamers (A-aptamer) bind to target cells, for the enrichment of targeted cells and strand displacement amplification (SDA), respectively. After the binding reaction, streptavidin (SA)-coated magnetic beads are added to form cell-magnetic bead-aptamer complexes that are collected for SDA amplification. Finally, the single strand amplicons are loaded onto the lateral flow strip for visual readout or hybridized to fluorophore-probe after displacement of quencher-labeled probe for fluorescent detection.-

#### **3. SDA product analysis-**

Detection of amplified product is critical in isothermal amplification of nucleic acids, which includes quantification of the final amplification product and monitoring of the product during the process of amplification. Hereafter, we introduce a variety of methods for the detection of nucleic acids, which include intercalating fluorescent dyes, fluorescent probes, lateral flow biosensors, and CRISPR/Cas system.-
