Microfluidic chip and isothermal amplification technologies for the detection of pathogenic nucleic acid

Abstract

The frequency of outbreaks of newly emerging infectious diseases has increased in recent years. The coronavirus disease 2019 (COVID-19) outbreak in late 2019 has caused a global pandemic, seriously endangering human health and social stability. Rapid detection of infectious disease pathogens is a key prerequisite for the early screening of cases and the reduction in transmission risk. Fluorescence quantitative polymerase chain reaction (qPCR) is currently the most commonly used pathogen detection method, but this method has high requirements in terms of operating staff, instrumentation, venues, and so forth. As a result, its application in the settings such as poorly conditioned communities and grassroots has been limited, and the detection needs of the first-line field cannot be met. The development of point-of-care testing (POCT) technology is of great practical significance for preventing and controlling infectious diseases. Isothermal amplification technology has advantages such as mild reaction conditions and low instrument dependence. It has a promising prospect in the development of POCT, combined with the advantages of high integration and portability of microfluidic chip technology. This study summarized the principles of several representative isothermal amplification techniques, as well as their advantages and disadvantages. Particularly, it reviewed the research progress on microfluidic chip–based recombinase polymerase isothermal amplification technology and highlighted future prospects.

Microfluidic systems for infectious disease diagnostics

“…In addition to immunoassay-based approaches, different types of nucleic acid amplification tests/technologies (NAAT), enabling highly specific and sensitive pathogen detection, are currently integrated with emerging microfluidic/biosensor diagnostic technologies. 83,97,98 A key method is polymerase chain reaction (PCR) requiring multiple temperature cycles to amplify DNA strands. Reverse transcription-PCR (RT-PCR) is utilized for amplification of viral RNA.…”

Evolution of the Probe-Based Loop-Mediated Isothermal Amplification (LAMP) Assays in Pathogen Detection

“…As a promising method for nucleic acid detection, LAMP has attracted more and more attention in the last two decades for POCT detection of animal and plant viruses, bacteria, fungi, mycoplasma, parasites, and other pathogens due to its high sensitivity, rapidity, and simplicity [6,19,26,79,80]. The major reason making the traditional LAMP methods inferior to qPCR assays and limiting their commercial and clinical application is their low specificity and accuracy, which are ascribed to non-specific amplification and/or the use of sequence-independent interferents (e.g., non-specific double-stranded DNA binding dyes) [15,35,77].…”

“…This technology can be simply performed in low-cost devices and has a fast processing time compared to qPCR, making it particularly suitable for point-of-care testing (POCT) [17][18][19]. After the pioneering development of the rolling circle amplification (RCA), various alternative approaches were developed to enable isothermal amplification, mainly including loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), helicase-dependent amplification (HDA), and so forth [20][21][22][23][24][25][26]. LAMP is one of the most frequently used isothermal amplification methods [16].…”

“…Diagnostics 2023, 13, x FOR PEER REVIEW 2 of 22 mal amplification, mainly including loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), helicase-dependent amplification (HDA), and so forth [20][21][22][23][24][25][26]. LAMP is one of the most frequently used isothermal amplification methods [16].…”

Isothermal amplification-based microfluidic devices for detecting foodborne pathogens: a review

“…Because isothermal amplification is carried out without precise thermal control, their applications are expanded in the integration with microfluidic devices to realize diagnostic tools. 14,15…”