Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are essential for all forms of life. Due to the versatile functions and broad applications of nucleic acids, methods for detecting nucleic acids from complex media have been developed. Among these methods, polymerase chain reaction (PCR) was the first and remains the most popular amplification technique for the amplification and detection of low-abundance nucleic acids. Although PCR has been widely used in various fields, it requires large and expensive thermal cycles, which limits PCR application to a large extent. Isothermal nucleic acid amplification has emerged as a promising alternative that rapidly and efficiently amplifies nucleic acid targets at constant temperature without the need for thermal cycling in PCR. This technique appears especially appealing owing to characteristics such as isothermal nature, simplicity, cost-effectiveness, robustness, sensitivity, specificity, and excellent amplification efficiency.
Isothermal nucleic acid amplification techniques employing various amplification mechanisms have been developed, such as transcription mediated amplification (TMA) or self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), signal-mediated amplification of RNA technology (SMART), strand displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification of DNA (LAMP), isothermal multiple displacement amplification (IMDA), helicase dependent amplification (HAD), single primer isothermal amplification (SPIA), and circular helicase-dependent amplification (cHDA). Most of these techniques have impressive sensitivity for detecting nucleic acids. The isothermal amplification methods operate at a uniform temperature and does not require variation during the process, thus eliminating the use of thermocyclers. In addition, real-time readings of amplification are possible with these methods, and amplified products can be detected by measuring turbidity or by visual inspection for color change. This capability eliminates the need for gel electrophoresis. Isothermal amplification can be integrated into simple and compact systems due to its low energy requirements and simplicity. The combination of isothermal amplification technology and nanotechnology has also attracted great attention. Isothermal amplification provides abundant nucleic acid materials for the construction of high-order nucleic acid nanostructures, nucleic acid template-metal nanostructures, and nucleic acid hydrogels, which has broad application prospects in biosensing, bioimaging, and nanomedicines. Isothermal amplification methods have evolved from originally test-tube based, to coupling with microfluidic chips, capillary platforms, and test strips. Moreover, isothermal amplification technology has been extended from detecting nucleic acids to detecting a wide range of targets, including proteins, cells, small molecules, and ions.
Isothermal amplification methods differ from one another in features such as the number of primers and enzymes, the temperature of amplification and template types used. These techniques also differ in their requirements for sample volume, specimen preparation and methods of amplification and detection. There are strengths and weakness to each of the amplification systems. Therefore, an appropriate isothermal nucleic acid amplification method should be selected according to the research needs.
Note: If you don't receive our verification email, do the following: