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.