**4. Discussion, conclusion, and future perspectives-**

The development of new methodologies in nucleic acid amplification is of great importance- in point of care diagnosis for research and public health. The SDA and its colleagues such as- RPA, LAMP, etc. have proved reliable application for this purpose. Nevertheless, SDA has- poorly achieved longer sequence amplification and a comparable amplification as for LAMP- toward turbidity measurement, but under stringent conditions, possesses higher sensitivity, specificity, and cost-efficiency, and can be multiplexed and reprogrammed for various- targets [58, 93–96]. But also, other isothermal amplification methods such as LAMP have- hardly shown multiplexing capacity [97–99] and are solely dye dependent for colorimetric- detection and quantification, which are target independent and nonspecific. Up to now, no- perfect method can overcome all shortcomings. However, probe-based lateral flow assay in- combination with SDA could provide robust multiplexing detection higher than their peer- antibodies [84]. For example, our group [15, 24, 36] and other researchers [3, 100, 101] demonstrated that SDA can be used to amplify short target recognition sequences, for example,- aptamers, after target binding and then integrated with lateral flow biosensors for analysis.- Therefore, preheat denaturation of templates, background signals, and unspecificity encountered during the use of long nucleic acid templates or the presence of DNA contaminants- are prevented [102, 103]. Furthermore, the lab-on chip approach, consisting of on-chip fixed- multiple analytes, could eliminate partially primer-dimerization frequently observed during- multiplexed isothermal amplification [104].-

DNA fluorescent probes were widely applied for singlex and multiplex detection of isothermal amplified nucleic acid targets. Notwithstanding, comparing with fluorescent probe detection of nucleic acids and other PCR-based techniques, CRISPR-Cas-based analysis of isothermal amplified products is fast, sensitive, and specific, allowing its on-site implementation. This technique is reprogrammable for the ultrasensitive and specific detection of various targets and is amenable to multiplexing by using their targeting Cas proteins and fluorescent labels [11, 13, 60]. Nevertheless, further studies are still needed to overcome some drawbacks of this technology. For example, the cost of enzymes and anti-fluorophore reporters still needs to be reduced. Moreover, the operability needs to be improved because the usage of more enzymes in one reaction requires complicated optimization for one-pot concurrent catalysis. In the lateral flow assay, we propose the combination of CRISPR-Cas with DNA capture probe technique for the cleaved and noncleaved reporters' detection, in order to replace the immunoassay approaches which are prone to cross reactivity. Furthermore, the sensitivity of this emerging diagnosis depends solely on target (s) nucleic acid (s) amplification. Thus, alternatively, one can develop an intrinsic signal amplification of reporters' signal by either cationic and fluorescent conjugated polymers or direct inhibition of fluorescent loss using fluorophore encapsulation. For signal amplification, the CRISPR system might be promoted by using other fluorescent-intensity-based nanostructures (e.g., quantum dots and silver nanostructures) and up conversion nanomaterials alongside with robust quenchers such as gold nanoparticles [105] and carbon nanomaterials (graphene oxides and single-walled carbon nanotubes) [106]. Additionally, the use of G-quadruplex-mediated catalysis for colorimetric detection of isothermal amplified amplicons or cleaved-CRISPR reporters could eliminate the need of more enzymes and thus allow conveniently the one-pot reaction toward simplified, cost-effective, specific, and sensitive on-site detection of nucleic acids.-
