Key words
Isothermal amplification; RAA; Respiratory virus; Rapid detection;
1.Introduction
Respiratory viruses can be transmitted through airborne as well as close contact, usually proliferate in the mucosal epithelium of the respiratory tract, causing localized infection of the respiratory tract or lesions of tissues and organs outside the respiratory tract1-3. Infected individuals always present with non-specific symptoms such as fever, cough, malaise and loss of smell, which in severe cases can lead to pneumonia, respiratory failure, multiple organ damage, and even shock or death4, 5. Acute respiratory diseases caused by respiratory viruses, such as COVID-19, have posed a serious threat to human health and social stability in the past decades6, 7. The respiratory syncytial virus, influenza A virus, influenza B virus, parainfluenza virus, and adenovirus are the most common respiratory viruses and can be easily and fast transmitted from person to person8, 9. Especially the COVID-19 outbreak in 2020, it is estimated that as of April 8, 2023, the global cumulative number of infections has exceeded 684 million and the cumulative number of deaths exceeded six million10. The epidemic is still spreading worldwide11. Early and accurate diagnosis are essential to control pandemics caused by respiratory viruses because of the non-specific symptoms and insidious transmission of asymptomatic infections12, 13. Therefore, rapid detection of respiratory viruses is of great importance.
Nucleic acid detection is still the gold standard for pathogen diagnosis. Polymerase chain reaction (PCR) is one of the most common methods for nucleic acid detection14. However, some disadvantages limit its widespread use, especially grassroots and field applications, such as the need for complex and time-consuming thermal cycling processes, expensive detection equipment, and specialized laboratory conditions15. Immunological assays such as enzyme-linked immunosorbent assay (ELISA) and colloidal gold assay are prone to false negatives due to their relatively low sensitivity14. Therefore, the development of sensitive, rapid and simple methods for detecting respiratory viruses is of great practical significance in early screening of cases and reducing the risk of transmission16.
In recent years, the continuous development of various isothermal amplification techniques has brought new opportunities for field detection17, 18. They could perform nucleic acid amplification at a constant temperature, and do not depend on thermal cycling instruments19. Several promising isothermal amplification methods are already available, but some of them require relatively high temperatures, long reaction times, or complex primer designs. For example, loop-mediated isothermal amplification (LAMP) requires 4-6 primers, leading to the failure to design appropriate primers for some pathogen genes, and a high working temperature of 65°C20, 21. Nucleic acid sequence-based amplification (NASBA) requires a longer amplification time (typically 90 min)22. In contrast, the recombinase aided amplification (RAA) method works successfully over a wide temperature range of 37 ~ 42°C using specific primer pairs, thus enabling amplification even at room temperature or human body temperature23, 24. RAA is considered to be a promising alternative to PCR for rapid nucleic acid detection25. The schematic diagram of the RAA amplification is shown in Figure 1. The recombinant enzyme in RAA reaction system, obtained from bacteria or fungi, can tightly bind to the primer DNA at room temperature to form an enzyme-primer polymer. When the primer searches for a complementary sequence on the template DNA, the recombinant enzyme opens the double-stranded structure of the template DNA with the aid of single-stranded DNA binding protein. Under the action of DNA polymerase, a new DNA complementary strand is formed, and the amplification product grows exponentially26. The detection results can be obtained in 20 minutes using fluorescent probes. RAA has the characteristics of simple operation, fast amplification speed, high sensitivity and specificity, and is very promising for application, which can meet the needs of not only the front-line field detection, but also the lack of conditions in the community, grassroots and other scenarios.