Viruses and clinical samples
CSFV (C vaccine strain), PCV2 (SX07 vaccine strain), PRRSV (TJM-F92 vaccine strain), FMDV (O/Mya98/XJ/2010 vaccine strain), PRV (Bartha-K61 vaccine strain), PPV (N vaccine strain), PEDV (CV777 vaccine strain), TGEV (H vaccine strain), PRoV (NX vaccine strain) were stored in our laboratory. The ASFV, APPV, BVDV-1, BVDV-2, BDV and PDCoV positive clinical samples were collected in the fields, confirmed by PCR/RT-PCR and gene sequencing, and stored in our laboratory.
A total of 509 clinical samples, including brain, lung, liver, spleen and lymph nodes for each naturally dead pig, were collected from different pig herds in Guangxi Province, Southern China from October 2018 to December 2020. All clinical samples were stored at -80℃ until used.
Primers and TaqMan probes
Three pairs of specific primers and TaqMan probes used for multiplex qRT-PCR assay were designed using software Primer Express 3.0 basing on the genomic sequences of ASFV (GenBank accession number NC_001659), CSFV (NC_002657) and APPV (KY624591), respectively, which amplified 79 bp fragment for ASFV p72 gene, 72 bp fragment for CSFV 5′UTR and 90 bp fragment for APPV 5′UTR, respectively. The detailed information of primers and probes were listed in Table 1.
Table 1 Primers and probes used for detection of ASFV, CSFV and APPV
Extraction of nucleic acid
All vaccine viruses and the pooled clinical tissue homogenates (20 %, W/V) were resuspended with phosphate buffer saline (PBS, pH7.2), vortexed and centrifuged at 12, 000× g at 4℃ for 5 min. Total RNA and DNA was extracted from the supernatants using MiniBEST RNA/DNA Extraction Kit Ver.5.0 (TaKaRa, Dalian, China) according to the manufacture's instructions, and stored at -80℃ until used.
Construction of standard plasmids
Total DNA was extracted from ASFV positive sample, and total RNA were extracted from CSFV vaccine and APPV positive sample and then reversed transcribed to cDNA. The target fragments of ASFV, CSFV and APPV were amplified by PCR using ASFV DNA and CSFV, APPV cDNA as templates. The amplicons were purified and cloned into pMD18-T vector (TaKaRa, Dalian, China) and transferred into E. coli DH5α competent cells (TaKaRa, Dalian, China). The positive clones were cultured at 37°C for 18 h-20 h and extracted by MiniBEST Plasmid Extraction Kit Ver.5.0 (TaKaRa, Dalian, China) for plasmid constructs. The plasmids were named as p-ASFV, p-CSFV and p-APPV, respectively, and stored at –80°C until used as standard plasmids.
The standard plasmids were quantified by ultraviolet absorbance at 260 nm and 280 nm with a NanoDrop Sectrophotometer (Thermo Fisher, USA). The exact copy numbers of plasmids were calculated using the following formula:
Plasmid copies/μL = (6.02 × 1023) ×(X ng/μL × 10-9)/plasmid length (bp) × 660
Optimization of the single qRT-PCR assay
The standard plasmids were 10-fold serially diluted from 2.52 × 109 copies/μL to 2.52 × 101 copies/μL （final reaction concentrations: from 2.52 × 108 copies/μL to 2.52 × 100 copies/μL） for optimizing the reaction conditions of the single qRT-PCR of ASFV, CSFV and APPV. A total volume of 20 μL contained: 2× One Step qRT-PCR Buffer III (TaKaRa, Dalian, China) 10 μL, Ex Taq HS (5 U/μL) (TaKaRa, Dalian, China) 0.4 μL, PrimeScript RT Enzyme Mix II (TaKaRa, Dalian, China) 0.4 μL, each primer 0.1-0.6 μL, probe 0.1-0.6 μL, plasmid template 2.0 μL and distilled water to a total volume of 20 μL. All reactions were amplified by an ABI QuantStudio™ 6 Real-time System (ABI, USA) and the amplification parameters were as follows: 42℃ for 5 min, followed by 95℃ for 10 s; then 40 cycles of 95℃ for 5 s and 59℃ for 34 s. The fluorescent signals were determined at the end of each cycle.
Optimization of the multiplex qRT-PCR assay
Based on the optical reaction conditions of the single qRT-PCR, the reaction conditions of the multiplex qRT-PCR, imcluding annealing temperature, primer concentrations, probe concentrations and so on, were further determined by orthogonal experiments.
A total volume of 20 μL contained: 10 μL of 2× One Step qRT-PCR Buffer III (TaKaRa, Dalian, China), 0.4 μL of Ex Taq HS (5 U/μL) (TaKaRa, Dalian, China), 0.4 μL of PrimeScript RT Enzyme Mix II (TaKaRa, Dalian, China) , 0.1-0.6 μL of the mixtures of primers and probes with different final concentrations, 2.0 μL of the three standard plasmids mixed in ratio of 1:1:1 with different final concentrations as templates, and sterilized distilled water to a final volume of 20 μL. The amplification parameters were as follows: 42℃ for 5 min, followed by incubation at 95℃ for 10 s; then 40 cycles of denaturation at 95℃ for 5 s and annealing and extension at 59℃ for 34 s. Finally, the fluorescent signals were determined at the end of each cycle. After amplification, a Ct value was assigned to each sample. The final concentrations of primers, probes and the amplification conditions were optimized to obtain the maximun ΔRn and minimal Ct using the standard plasmids of different dilutions as template.
Specificity analysis of the multiplex qRT-PCR
The DNA or RNA of ASFV, CSFV, APPV, PCV2, PRV, PRRSV, FMDV, PEDV, TGEV, PRoV, PDCoV, BVDV-1, BVDV-2 and BDV were used as templates of the developed multiplex qRT-PCR to verify the specificity of the assay.
Sensitivity analysis of the multiplex qRT-PCR
The standard plasmids of p-ASFV, p-CSFV and p-APPV were 10-fold serially diluted from 2.52 × 108 copies/μL to 2.52 × 100 copies/μL （final reaction concentrations: from 2.52 × 107 copies/μL to 2.52 × 10-1 copies/μL）and used as templates for the multiplex qRT-PCR to determine the sensitivity of the assay.
Repeatability analysis of the multiplex qRT-PCR
The standard plasmids of p-ASFV, p-CSFV and p-APPV were 10-fold serially diluted from 2.52 × 108 copies/μL to 2.52 × 100 copies/μL, and the concentrations of 2.52×108 copies/μL, 2.52×106 copies/μL and 2.52×104 copies/μL （final reaction concentrations: 2.52×107 copies/μL, 2.52×105 copies/μL and 2.52×103 copies/μL）were used as templates for the developed multiplex qRT-PCR. The coefficients of variation of the intra- and inter-assay were determined to evaluate the repeatability of the assay.
Detection of clinical samples by the multiplex qRT-PCR
A total of 509 clinical samples were collected from pig farms in Guangxi Province, Southern China from October 2018 to December 2020. The total RNA and DNA were extracted from 20 % tissue supernatants using MiniBEST RNA/DNA Extraction Kit Ver.5.0 (TaKaRa, Dalian, China) and were detected by the developed multiplex qRT-PCR for ASFV, CSFV and APPV.
Phylogenetic analysis based on ASFV p72 gene
Twenty-one samples were selected randomly from the positive samples of ASFV to amplify partial p72 gene using a pair of primers (P72-U: 5'-GGCACAAGTTCGGACATGT-3', P72-D: 5'-GTACTGTAACGCAGCACAG-3') as previously described . The PCR products were purified, ligated to pMD-18T vector (TaKaRa, Dalian, China) and transferred to E. coli. DH5α competent cells. The positive clones were selected and sequenced (TaKaRa, Dalian, China), and the acquired sequences were edited by the EditSeq program of the DNAstar software, and aligned with the reference strains retrieved from GenBank using Clustal W. Phylogenetic reconstruction was conducted using the Maximum-Likelihood algorithm method (T92+G). Phylogenetic tree reliability was supported using the Kimura distances and a bootstrap method with 1000 replications.