4. Discussion
In
the past decades, frequent outbreaks of infectious diseases caused by
respiratory viruses have posed a great threat to human health. In order
to prevent and control infectious disease epidemics as soon as possible,
rapid and accurate diagnostic methods are essential to identify the
pathogens. The use of PCR is limited by the need for complex and
time-consuming thermal cycling processes, expensive testing equipment
and specialized laboratory conditions, so it is not suitable for
grassroots and resource-poor areas. In this work, we established a
simple, rapid, highly sensitive and specific assay based on fluorescent
RT-RAA for the detection of six respiratory viruses (RSVA, Flu A, Flu B,
HPIV, SARS-CoV-2 and ADV), each of which could be completed within 20
min at a mild reaction temperature of 39°C. The sensitivity of the RAA
assay was tested at 102 copies/mL for RSVA,
102 copies/mL for HPIV, 102copies/mL for Flu A, 103 copies/mL for Flu B,
102 copies/mL for SARS-CoV-2, and
101 copies/mL for ADV, and was confirmed by
cross-testing with 21 different pathogen nucleic acids with good
specificity. Although the results of parallel testing of pharyngeal swab
samples by the RAA method were consistent with those of RT-qPCR, the
results obtained by the RAA method within 20 min were more time-saving
compared with the 1.5 h required by PCR, because the reaction conditions
were mild. Moreover, the required equipment by RT-RAA in this work was
more portable (only 2 kg) than that usually required by RT-PCR, and the
RT-RAA detector had a built-in battery, which did not require additional
power supply and could be carried to the field for testing.
In addition, different from PCR, which requires different heating and
cooling processes for the amplification of different pathogenic nucleic
acids, RT-assay established in this work can achieve the amplification
and detection of six respiratory virus nucleic acids using the same
thermostatic procedure. Therefore, in field detection, 16 separate
channels of RT-RAA portable detector can be used to flexibly choose
single pathogen detection or multiple pathogen detection for test
samples. For example, 14 samples (in addition to negative and positive
controls) can be tested for single pathogen, or the same sample can be
added to different channels for multiple detection of 1 to 6 respiratory
viruses at the same time.