2.Materials and methods

Primers and probes design

By retrieving the database of Global Initiative on Sharing All Influenza Data (GISAID), a total of 254 swine H1N1 viruses during 2009-2019 and all the 60 swine H3N2 viruses in China (up to 1 Oct, 2019) were downloaded for sequence alignment via MEGA (version 6.06) software. Next, suitable H1 (Fig.1 A) and H3 (Fig.1 B) primer-probe sets targeting the conserved HA gene regions were designed and validated by Primer Express 3.0 software, respectively. The primers and probes used in the study were as follows: H1 forward primer, GGCTAYCATGCBAACAATTC; H1 reverse primer, TGGGTTGCCMAGGATCCA; H1 probe, FAM-ACAGACACTGTMGACACA-MGB; H3 forward primer, GAAATGGGAAAAGCTCAATAATGA; H3 reverse primer, ATTTCTCATYCCTGTTGCCAA; H3 probe, HEX-CCAAATGGAAGCATT-MGB. The amplification length of the H1 and H3 primer-probe sets were 179 bp and 184 bp, respectively.

Reaction composition and thermal cycling

HiScript II U+ One Step qRT-PCR Probe Kit (Vazyme Co., Nanjing, China) was used in our H1 and H3 duplex RRT-PCR assay to achieve the effect of reverse transcription and quantitation in one reaction tube. The H1 and H3 primers and probes were all synthesized by Tsingke Biological Technology Corporation (Nanjing, China). The RRT-PCR assay was operated in a 20μL reaction mixture which contains 2x One Step PCR Mix (Vazyme) 10μL, Enzyme Mix (Vazyme) 1μL, both H1 and H3 probes (10μM) each 0.4μL, both H1 and H3 forward and reverse primers (10μM) each 0.8μL, RNA sample 4μL, 50x ROX 0.4μL and RNase-free water 0.6μL. Thermal cycling of the RRT-PCR was performed in an ABI 7500 FAST real-time PCR machine (Applied biosystems). The temperature control steps were as follows. Step 1: 55℃ for 15min of reverse transcription, step 2: 95℃ for 30s, step 3: 95℃ for 30s and then 60℃ for 38s with quantitation to maintain 40 cycles.

Standard plasmid for RRT-PCR sensitivity and repeatability determination

Three SIVs of F15, JS38 and SZ14 respectively from EA H1N1, Pdm09 H1N1 and HL H3N2 lineages were used to amplify the HA genes. Each amplified HA gene was then inserted into the pHW2000 vector to construct the plasmid for standard curve plotting and repeatability test of the RRT-PCR assay. In particular, the plasmids mixture was divided into two different sets to represent the duplex detection of either EA H1N1 or Pdm09 H1N1 with HL H3N2, one set contained F15-pHW2000 and SZ14-pHW2000 while the other included JS38-pHW2000 and SZ14-pHW2000. Finally, two plasmid sets were serially diluted to 500000, 50000, 5000, 500, 50, 5 copies/μL to perform duplex detection for drawing standard curves.
For testing repeatability, 10-fold concentrations from 50000~50 copies/μL of F15-pHW2000 and SZ14-pHW2000 plasmid mixture were selected to conduct inter- and intra-comparison. Specifically, triplicates of the 4 dilutions of plasmids were performed on the same plate within a run while each reactions were respectively repeated on 3 separate days between runs of the duplex RRT-PCR assay.

Virus strain for sensitivity and specificity assay

9 strains of SIV isolated by our laboratory during 2010~2019, including 4 EA H1N1, 3 Pdm09 H1N1 and 2 HL H3N2 were used in the study (Table.1).18,19 The viruses were all propagated in 9-day-old SPF chicken eggs and then the infected allantoic fluid was harvested to serve as viral seeds. And the value of 50% embryo infectious dose (EID50) was determined for each SIV to express the detection limit of the RRT-PCR method.
After evaluating the conservativeness of the designed primer-probe sets via the Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), some commonly circulated porcine viruses in China containing classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV2), porcine epidemic diarrhea virus (PEDV) and porcine parvovirus (PPV) were also applied to measure the specificity of the RRT-PCR method in clinical application.

Animal experiment

6-week-old female BALB/c mice (n=20) provided by the Laboratory Animal Center of Yangzhou University were intranasally inoculated with the H1N1 subtype SIV strain JS285 at the dosage of 105.0EID50. Every 5 infected mice were humanely euthanized on 1, 3, 5 and 7 days post inoculation (dpi), and the lungs were collected to serve as experimental samples for verifying the detection accuracy of the duplex RRT-PCR method as compared with traditional virus isolation. The animal experiment protocol was approved by Jiangsu Province Administrative Committee for Laboratory Animals (approved NO. SYXK-SU-2017-0044), and complied with the guidelines of Jiangsu laboratory animal welfare and ethics of Jiangsu Administrative Committee of Laboratory Animals.

Application of RRT-PCR in SIV epidemiological surveillance

Swine nasal swabs collecting from some slaughter-house in Jiangsu province monthly from October to December, 2019 were treated as the clinical specimen to test the application of the duplex RRT-PCR method in pathogenic surveillance. These swabs immersing in dulbecco’s modified eagle medium (DMEM) were packaged appropriately on ice and transported to the laboratory as soon as possible.

Virus isolation

Homogenated mice lung tissues and filtered swine nasal swabs were inoculated into the allantoic cavities of 9-day-old SPF embryonated chicken eggs. After incubation of 96h, the allantoic fluid were collected for hemagglutinin (HA) assay to determine the potential presence of SIV. And the subtype of the HA positive samples were defined by sequencing of the PCR amplicons with primers according to Hoffman et al.20

RNA extraction

An EASY spin tissue/cell RNA rapid extraction kit (Yuanpinghao, Beijing, China) was used to extract RNA. According to the manufacturer’s instructions, RNA was prepared from 350μL virus cultures or specimens, and eluted with 30μL DEPC treated water.