E z n a viral dna kit

According to the manufacturer’s protocols, total DNA was extracted from 250-mg fecal samples using the E.Z.N.A. viral DNA kit (Omega Bio-Tek, Norcross, GA, USA). High-quality DNA samples (optical density at 260/280 nm [OD_260/280] of ~1.8 to 2.2 and OD_260/230 of ≥2.0) were used to construct the sequencing library. The DNA was amplified before library preparation. Metagenomic libraries were prepared using the TruSeq Nano DNA sample preparation kit from Illumina (San Diego, CA), using 1 μg of total DNA. Illumina’s protocol was followed for DNA end repair, A-base addition, and the ligation of Illumina-indexed adaptors. The libraries were sized using 2% low-range ultra-agarose for selecting DNA target fragments of ~400 bp, followed by PCR amplification using Phusion DNA polymerase (New England BioLabs [NEB]) for 15 cycles. Metagenomic sequencing was performed by Shanghai Biozeron Biotechnology Co., Ltd. (Shanghai, China). All samples were sequenced in paired-end 150-bp mode on a next-generation sequencing platform.

The specific primer pair for qPCR was designed using Oligo 6.0 and the EHV-1 glycoprotein B (gB) gene sequence (GenBank accession number: M36298). The primer pair (forward, 5′-GCCATACGTCCCTGTCCGACAA-3; reverse, 5′-CCTCCACCTCCTCGACGATGC-3′) yielded a product of 302 bases (nt. 1,207 to 1,509 EHV-1 sequences) under PCR cycle conditions of 95°C for 2 min, 95°C for 5 s, 60°C for 30 s, 95°C for 15 s, 60°C for 1 min, 95°C for 30 s, and 60°C for 15 s. RK-13 cells were seeded in 6-well plates (1 × 10⁵ cells/well), treated with 10 μM BBM for 8 h at 37°C, and infected with a neuropathogenic strain of EHV-1 (YM2019 kindly provided by Prof. Duoliang Ran (21 ), GenBank: MT063054.1) at an MOI of 0.7. After 4 h, the media was changed for fresh DMEM. At 0, 12, 24, 36, and 48 h later, suspension DNA was extracted for cells provided with the E.Z.N.A^® Viral DNA Kit (D3892-01, Omega Bio-Tek, the United States), and quantified to 6.07 × 10⁵ copies/mL using a QuantiNova SYBR Green PCR Kit (No. 208054) and 7,500 Fast Real-Time PCR software (22 (link)).

Tissue samples were homogenized and diluted with sterile PBS, followed by centrifugation at 6000 × g for 10 min at 4 °C to remove residual tissue debris. Viral nucleic acid was extracted using TRIpure Reagent (Aidlab, Beijing, China) or E.Z.N.A.^® Viral DNA Kit (Omega Bio-tek, Georgia, USA) in accordance with the manufacturer’s protocol.
A series of primers (Additional file 3: Table S1) were designed to detect pathogens that can cause CT in piglets or other common clinical pathogens, including APPV, CSFV, lateral-shaking inducing neurodegenerative agent (Linda) virus, porcine teschovirus (PTV), Japanese encephalitis virus (JEV), Seneca Valley virus (SVV), porcine sapelovirus (PSV), foot and mouth disease virus (FMDV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), and porcine pseudorabies virus (PRV) using methods as previously described [3 (link), 16 (link)–19 (link)].

In 2022, lymph samples of piglets with respiratory diseases and emaciation were collected from a large-scale pig farm in Guangxi. Appropriate tissue was homogenized and diluted with sterile PBS, freeze-thawed twice at -80℃, and centrifuged at 9,000 rpm for 10 min at 4℃ to collect the supernatant. DNA was extracted using an E.Z.N.A.® Viral DNA Kit (Omega Bio-tek, Georgia, USA) following the manufacturer’s protocol. RNA was extracted using TRIzol reagent in accordance with the manufacturer’s protocol instructions. A series of primers (Additional fle 1: Table S1) were designed to detect pathogens, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine pseudorabies virus (PRV), porcine circovirus type 3 (PCV3), porcine circovirus type 4 (PCV4) using methods as previously described [14 (link)–18 ]. And the amplification of DNA/cDNA was performed using the following conditions: (i) 95 °C for 3 min, (ii) up to 32 cycles of 95 °C for 15 s, 56 °C 30 s, and 72 °C for 20 s, (iii) 72 °C for 10 min. A single positive PCV2 was determined by the above tests for virus isolation in the next step.

Blood samples, collected from the orbital vein, were taken from 20 mice. 10 Balb/c mice and 8 ICR mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd, two ICR mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animal experiments were approved by the Institutional Animal Care and Ethics Committee of Nanjing Agricultural University (Approval No. IACECNAU‐20100902). DNA from 200 μl of serum was extracted using an E.Z.N.A. viral DNA kit (Omega Bio‐tek, Inc) according to manufacturer's instructions. DNA was stored at −20°C for further use.

To detect the viral load of PCV2 in infected mice. The pEASY-Blunt-PCV2 plasmid was used as the standard plasmid to draw a standard curve, and the copy number of PCV2 was calculated using the standard curve. Various tissues of the infected mice were homogenized and diluted with sterile PBS, DNA was extracted using E.Z.N.A.® Viral DNA Kit (Omega Bio-tek, Georgia, USA) in accordance with the manufacturer’s protocol. Real-time quantitative PCR (qPCR) was carried out in the ViiA™ 7 Real-Time PCR System (Applied Biosytems, Grand Island, NY, USA) system with Go Taq® G2 Hot Start Polymerase (Promega (Beijing) Biotech Co., Ltd, Beijing, China.), according to the manufacturer’s instructions.