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.