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.