4.Discussion
In this study, an one-step duplex RRT-PCR with high sensitivity,
specificity and repeatability was successfully established and evaluated
for detection of H1 and H3 subtype SIVs. As revealed by relevant
epidemiological investigations and the recorded items in influenza virus
databases, EA H1N1 and Pdm09 H1N1 were the main circulating lineages of
H1 subtype SIV in the recent decade.19,21-23Therefore, we desired to detect either the two H1 lineages in our
developed RRT-PCR assay by design of a single primer-probe set targeting
to the highly conserved regions of both EA H1N1 and Pdm09 H1N1. Although
the dominantly prevalent status of EA H1N1 in China has been more and
more evidently since 2011, part or even the whole set of internal genes
of Pdm09 H1N1 were yet found to be embeded in epidemic strains of H1 and
H3 subtype SIVs8,24. Hence, the possibility of the
risk that Pdm09 H1N1 virus may revival again in pig herds should not be
ignored, and the suitable detection methods are indispensably required.
Before applying the duplex RRT-PCR method in detection of clinical swab
samples, we initially evaluated the test accuracy in experimental
samples. Among the overall 18 mice lung samples that had been determined
positive by the RRT-PCR, 17 were isolated successfully by inoculating
into chicken embryos. And the coincidence rate between the two methods
was nearly 94.44%. Of note, 2 specimens on 1 dpi failed to be detected
as HA positive until a second blind-passage in SPF chicken embryos,
which alternatively indicated that the RRT-PCR might be more sensitive
than virus isolation. Subsequently, the duplex RRT-PCR method was
further applied for SIV surveillance in swine nasal swabs from some
slaughterhouse in China in 2019. And the three-month data from October
to December revealed that H1 and H3 subtype SIVs co-circulated in each
month in the investigated area. Particularly, H1 possessed an obviously
higher overall positive rate than H3 subtype SIV, which was generally
consistent with the results reported by some other research groups in
China.8,21
As possessing higher sensitivity and shorter testing time over
traditional virus isolation, more and more RRT-PCR methods have been
developed for SIV detection. For example, Slomka et al11 had reported two independent RRT-PCR assays
targeting the M gene or the HA gene to detect established SIVs or Pdm09
H1N1 in European pig populations, respectively. Additionally, to
differentiate the Pdm09 H1N1 from other influenza viruses containing
classical swine H1, EA H1, HL H1/H3 and avian H9N2 in swine, Hiromoto et
al 25 had designed six pairs of HA gene primer-probe
sets that were respectively applied in each singleplex RRT-PCR assay.
Besides, Malliga et al established two tetraplex RRT-PCR methods for the
simultaneous detection of common M, H1, H3 SIV genes and N2 SIV genes or
H5 genes from Eurasian highly pathogenic avian influenza
viruses.26 Although those RRT-PCR for SIV detection
are documented with different advantages, they could hardly
differentiate the prevalent lineages of EA H1N1, Pdm09 H1N1 and HL H3N2
in China within a single multiplex reaction.
Our duplex RRT-PCR employed two HA specific primer-probe sets to detect
the H1 and H3 subtype SIVs at the same time, especially that the H1
primers and fluorescent probe could simultaneously match the EA H1N1 and
Pdm09 H1N1. The detection limit of the method could reach 5 copies/μL
nucleic acids of HA gene or 0.21~4.2
EID50 of SIV per reaction, and the CV was lower than 3%
in both intra- and inter-assays which indicated good repeatability.
Besides, no mismatch with other HA subtypes influenza viruses via the
Primer-BLAST search, and no cross reaction with other swine viruses that
commonly existed in China further emphasized the good specificity of the
method. Therefore, this duplex RRT-PCR for H1 and H3 subtype SIV
detection not merely presented high specificity, sensitivity and
repeatability but also showed high detection precision rate in the
comparison with virus isolation, which indicated a bright application
prospect in serving the method as an effective tool for SIV surveillance
and offering instructive information for SIV isolation.