Viral dna kit

NGPV loads in the liver, spleen, and cloacal swabs were quantified using quantitative real-time PCR (qPCR). The primer pairs targeting the NGPV NS helicase gene SF3 domain were designed as follows: forward, 5′-TACAATGGAACACAGAATWCC-3′ and reverse, 5′-TCAGACACAACAGGAACWA-3′. Viral DNA extracted from tissue or cloacal swab samples using a viral DNA kit (Omega Bio-Tek, Norcross, GA, USA) was used as the template for qPCR. The qPCR reaction system was 20 µL in volume, containing 0.5 µL forward primer, 0.5 µL reverse primer, 10 µL AceQ Universal SYBR qPCR Master Mix (Vazyme Inc., Nanjing, China), 2.0 µL of the template DNA, and 7.0 µL ddH₂O. The cycling conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s.

To quantify the amount of HBV, HBV DNA was extracted from the patient’s sera or culture medium using a Viral DNA kit (Omegabiotek, D3892) according to the manufacturer’s instruction. qPCR was performed as described previously. Briefly, primers used for amplification were: GGGAGGAGATTAGGTTAA and GGCAAAAACGAGAGTAACTC. HBV 1.3-mer WT replicon (Addgene, plasmid#65459) was used as standards. Serially diluted standards were processed in parallel with samples to generate a standard curve for quantification.

Total RNA was extracted using the cell total RNA Isolation Kit (FORGENE) and then reverse transcribed with the PrimeScript RT reagent Kit (RR047A, Takara, Shiga, Japan). RT-qPCR was performed in triplicate using SYBR premix Ex TaqII Kit (RR820, TaKaRa). The mRNA level was quantified by measuring the cycle threshold (CT) value. Data was normalized to the level of the control gene encoding GAPDH. The ΔΔCt method was used to measure the expression levels of target genes. According to the concentration of pMD19-T-gB and the corresponding CT value, the standard curve was established and the calculation formula of PRV copies was obtained. PRV DNA was extracted from different viral fluids by E.Z.N.A. Viral DNA Kit (Omega Bio-tek, Doraville, GA, United States). The PRV copies were detected by RT-qPCR using the extracted virus DNA. The primers are listed in Supplementary Table S2.

Virus attachment capability was assessed indirectly through quantification of viral genomic DNA of the viruses that bound on the surface of the selected cells after wash off of the unbonded viruses within 1 hpi as described previously [28 (link)]. Cells were infected with ASFV (MOI = 5) for 15 min, 30 min and 1 h at 4 °C. The cells were then washed with ice-cold PBS three times, and the ASFV genomic DNA was extracted from the washed cells using E.A.N.A. Viral DNA Kit (Omega Bio-tek, Norcross, GA, USA) was utilized following the manufacturer’s instructions. The quantification of ASFV genomic DNA was performed with real-time PCR using AceQ qPCR Probe Master Mix (Vazyme, Shanghai, China) with primers targeting ASFV B646L gene (Table 2).

Viral genomic DNA was extracted from serum or tissues using a Viral DNA Kit (Omega Bio-Tek, Inc., Norcross, GA, USA) according to the manufacturer’s protocol. To determine the EHV-8 replication in mice, a real-time PCR assay was used to detect EHV-8 in serum based on part ORF70 gene amplification with G1 F and R primers (Table 1). The amplicons were inserted into the pMD18-T vector to generate recombinant plasmids, which were used to develop the real-time PCR assay as previously described [19 (link)]. Simultaneously, PCR was performed with G2 F and R primers (Table 1) to detect the EHV-8 distribution in mice tissues at 2, 4, and 6 dpi.
The virus titers in tissues of EHV-8-infected mice were further determined in RK-13 cells. Briefly, three mice from group 2 were sacrificed at 6 dpi, and their liver, spleen, lung, kidney, and brain were collected. These tissues (0.1 g) mixed with PBS (1 mL) were crushed, homogenized, then frozen and thawed 3 times. After that, the supernatant was collected from different tissues, filtered through a 0.22 µm syringe filter, and tittered in RK-13 cells using the Reed–Muench method described previously [12 (link)].

To explore the proliferation capability of AaeDV, AalDV-3 and recombinant virus rAaeDV-210 in mosquito cells, C6/36 and Aag2 cells were seeded in 24-well plates (C6/36 cells: 1 × 10⁵ cells/well, Aag2 cells: 1 × 10⁶ cells/well) and incubated with 1 ml of media containing virus at a final concentration of 3.00 × 10⁹ genome equivalents per ml (geq/ml) at 28 °C. After incubation for 24 h, the medium was removed, and the cells were washed 3 times with fresh RPMI-1640 medium (Schneider’s Drosophila medium for Aag2 cells) to remove residual virus. Then, 1 ml of fresh medium was added, and the cells were returned to the incubator for regular culture.
Viral titers in the cell culture supernatant and in the cells themselves were determined by quantitative real-time PCR (qPCR). First, to determine the titer of the extracellular viruses, samples from the cell culture were collected at serial time points post-infection [0, 2, 4, 6, 8 and 10 days post-infection (dpi)] and then centrifuged for 10 min at 1000× g. Supernatants were used to extract viral genomic DNA using a Viral DNA Kit (Omega Biotek, Norcross, GA, USA). To determine the titer of the intracellular virus, the supernatants were removed, and the infected cells were washed 3 times with 1× PBS. The cells were collected at serial time points post-infection (0, 2, 4, 6, 8 and 10 dpi); the genomic DNA was then isolated for qPCR.

Lungs (0.1 g) from different groups mixed with PBS (1 mL) were crushed, homogenized, and then frozen and thawed 3 times. After that, the supernatant was collected to extract viral genomic DNA using a Viral DNA Kit (Omega Bio-Tek, Inc., Norcross, GA, USA) according to the manufacturer’s protocol. To determine the EqHV-8 replication in the lungs, a qPCR assay was used to detect EqHV-8 based on the above methods. Finally, viral loads were calculated in the lungs by absolute quantification.