3.Result
3.1 Standard curves and amplification plots of
RRT-PCR
The amplification plots and standard curves for the two reaction sets
including plasmids of EA H1N1 and HL H3N2 (Fig.2, Panel A) or plasmids
of Pdm H1N1 and HL H3N2 (Fig.2, Panel B) were determined under the
circumstance of serially diluting each plasmid into 500000, 50000, 5000,
500, 50 and 5 copies/μL, respectively. As shown from the amplification
plots in Fig.2, the detection limit for either duplex detection of EA or
Pdm09 H1N1 and HL H3N2 plasmids was 5 copies/μL. And the standard curves
indicated that the linear correlation for EA H1N1 and HL H3N2 group
were y=-3.38x+36.106 (Efficiency
97.640%, R2=0.999) and y=-3.335x+ 37.406 (Efficiency
99.470%, R2=0.999) while for Pdm09 H1N1 and HL H3N2
group were y=-3.301x+35.933 (Efficiency 100.889%,
R2=0.997) and y=-3.301x+35.977 (Efficiency 100.885%,
R2=0.996), respectively.
3.2 Repeatability evaluation of
RRT-PCR
For evaluating the repeatability of the RRT-PCR assay, the plasmid
mixture of F15-PHW2000 and SZ14-PHW2000 was serially diluted to 50000,
5000, 500, 50 copies/μL and then used to perform the intra- and
inter-comparison. As shown in Table 2, the coefficient values (CV) for
the duplex detection of H1 and H3 subtype SIV were both < 3%
between runs or with a run, which suggested a high repeatability of the
method.
3.3 Detection limit of different virus
strains
By using different HA subtypes or lineages of SIV strains, the detection
limit of the duplex RRT-PCR was also calculated as expressed in
EID50. As displayed in Fig.3, the lowest detection
concentrations ranged from 0.21 to 4.2 EID50 for the 4
EA H1N1 viruses (F15, SG03, ZG14, ZG16) and from 2.1 to 3.2
EID50 for the 3 Pdm09 H1N1 viruses (JS38, JS48, JS285),
respectively. Whereas for the 2 HL H3N2 viruses (SZ14, YC44), the
positive threshold were both 0.24 EID50.
3.4 Specificity evaluation of
RRT-PCR
The specificity of the designed primers and probes for the duplex
RRT-PCR were initially evaluated through the analysis tool of
Primer-BLAST. As shown in Table.3, the H1 primer-probe set matched
mainly H1N1 and secondly H1N2 and then some H1N3-N6 influenza viruses
while the H3 primer-probe set matched predominantly H3N2 and some H3N1
or H3N2 viruses, respectively. Evidently, no HA subtypes other than H1
or H3 could be searched to coincident with the specific primers and
probes.
Subsequently, the specificity of the RRT-PCR assay in clinical
application was further measured with some other commonly-existed swine
pathogens in China, including CSFV, PRV, PRRSV, PCV2, PEDV and PPV. As
obviously shown in Supplementary Fig.S1, just the positive control of H1
and H3 SIVs displayed amplification curves in their individual
fluorescence channel (FAM and HEX), whereas the other 6 swine viruses
were all detected as negative.
3.5 Detection accuracy of the RRT-PCR in experimental
samples
To compare the detection accuracy of the duplex RRT-PCR method with
classical virus isolation, lungs from JS285(H1N1) infected mice were
treated for RRT-PCR detection and embryo inoculation, respectively.
During the 7-day trial period, 5 lung specimens were collected every
other day since 1 dpi except that just 3 samples were obtained on 7 dpi
due to the death of 2 mice. As shown in Fig.4, each of the 13 lung
samples on 3, 5 and 7 dpi were tested positive via both virus isolation
and the RRT-PCR. However, in terms of the 5 samples on 1 dpi, although
they were all identified positive by the RRT-PCR, only 4 samples were
successfully isolated from egg inoculation with 2 at the first
inoculation and the other 2 after 3 consecutive blind passages.
3.6 Application of the RRT-PCR in SIV
surveillance
A total of 166 swine nasal swabs collecting monthly in October (n=54),
November (n=56) and December (n=56), 2019 were used for SIV surveillance
by applying the RRT-PCR method. As shown in Fig.5, both H1 and H3
subtype SIVs were detected in each month. Specifically, 5, 16 and 5
samples were H1 positive while 7, 7 and 2 samples were H3 positive in
October, November and December, respectively. Thus, the corresponding
positive percentages were 9.26%, 28.57%, 8.92% for H1 and 12.96%,
12.50%, 3.57% for H3. In addition, the overall prevalent rate of H1
(15.66%) was relatively higher than that of H3 (9.64%) subtype SIV.