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.