Recent advances in cascade isothermal amplification techniques for ultra-sensitive nucleic acid detection

Combination of nucleic acid amplification and CRISPR/Cas technology in pathogen detection

“…PCR is still the most mature technology in detection, and isothermal nucleic acid amplification technology is becoming more mature in microbial detection, which can compensate for PCR’s dependence on application scenarios and instrumentation, and has the characteristics of rapid, sensitive and convenient detection, and is especially suitable for rapid on-site detection in remote areas ( Gill and Ghaemi, 2008 ). It is the main direction of future development in the field of pathogen nucleic acid detection ( Deng and Gao, 2015 ; Jiang et al, 2023 ). However, compared with traditional PCR, primer design in isothermal nucleic acid amplification is complicated and difficult, lacks appropriate design software, requires screening and optimization of reasonable primers ( Mayboroda et al, 2018 ), and is more prone to false-positive results due to aerosol contamination or non-specific amplification, and there is still a certain gap between detection sensitivity and qPCR.…”

Target-triggered dual signal amplification based on HCR-enhanced nanozyme activity for the sensitive visual detection of Escherichia coli

“…At present, traditional methods for detecting bacteria mainly rely on bacterial culture, 5,6 polymerase chain reactions (PCR), 7,8 and enzyme-linked immunosorbent assays (ELISA). 9,10 In detail, the method of bacterial culture is to isolate the pure species of the sample on a plate medium, and then identify the bacteria according to the appearance characteristics of the size, color, shape, and transparency of the bacterial growth colony.…”

Rapid detection of Mycobacterium tuberculosis in sputum using CRISPR-Cas12b combined with cross-priming amplification in a single reaction

“…By contrast, isothermal amplification is a straightforward, rapid, and efficient nucleic acid amplification process that can substitute expensive thermocyclers with a water bath, significantly streamlining the nucleic acid amplification workflow. With the emergence of various isothermal polymerases, a range of enzyme-mediated isothermal nucleic acid amplification techniques have surfaced, including loop-mediated isothermal amplification (LAMP) ( 14 ), recombinase polymerase amplification (RPA), and cross-priming amplification (CPA) ( 12 , 15 ). CPA relies on a chain-replacing DNA polymerase and is capable of exponential amplification of DNA target sequences, guided by multiple cross-linked primers (Ustar Biotechnologies, Hangzhou, China).…”

“…Although isothermal amplification has overcome temperature control challenges, it still faces limitations related to false positives, nucleic acid sequence compatibility, and signal amplification capacity. Fortunately, by integrating isothermal amplification with the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) enzymes to achieve cascade bioconversion, these limitations can be effectively addressed ( 15 ).…”

“…This cleavage generates detectable fluorescent signals. The highly efficient trans-cleavage acts as a superior signal amplification method, elevating sensitivity by approximately two to three orders of magnitude ( 15 ). The combination of the CRISPR system with isothermal methods for pathogen detection has resulted in the development of various CRISPR-based nucleic acid detection techniques ( 18 , 19 ).…”