Primestar gxl kit

Three regions were amplified using Primestar GXL kit with GC buffer (Takara Bio Inc., Japan), in 25 μL final volume with 1 μL of purified ASFV DNA according to the manufacturer’s instructions. The amplification was performed using the following PCR program: 98 °C 30 s, 30 × (98 °C 10 s, 60 °C 15 s, 68 °C 1 min), 68 °C 2 min. The primer pairs used for the amplification of the different fragments are listed in Table 1. The same primers were used for Sanger sequencing by BaseClear B.V. (Leiden, Netherlands). Sequences of the amplicons were well defined from both directions upstream of the poly C/G on each fragment, while downstream of the C/G tracts sequence slippage was detected in each case. The size of the poly C/G tracts and the actual sequence around them were determined by comparing and analyzing the reads of the opposing strands.

Genomic DNA of 14 open reading frames (ORFs) in the candidate region were amplified in ZS9 and yvl using gene-specific primers (Table S2) by using a PrimeSTAR GXL Kit (Takara, Japan) and cloned into the PBI121 Vector using ClonExpress II One Step Cloning Kit C112-01 (Vazyme). The recombinant plasmids were transformed into DH5α competent cells for sequencing. Multiple sequence alignment was performed with Clustal Omega online software (http://www.ebi.ac.uk/Tools/msa/clustalo/) and visualized by Sequence Manipulation Suite: Color Align Conservation (http://www.bioinformatics.org/sms2/color_align_cons.html).

The identification of specimens identified as being infected with BTV was confirmed by amplification of the cytochrome c oxidase subunit 1 (cox1) gene using a method modified from Duan et al. [19 (link)]. Briefly, 5.5 μl of DNA was added to 14.5 μl of reaction solution prepared using the PrimeSTAR® GXL kit (Takara) and primers BC1culicFm and JerR2m of Bellis et al. [25 ]. PCR cycling conditions were: 95 °C, 2 min; then 95 °C/10 s, 45 °C/10 s, 68 °C/50 s for 30 cycles; with a final extension at 68 °C for 30 s, followed by incubation at 4 °C. Subsequently, 10 μl of fresh 1× PCR solution was added to each tube and a second round of PCR was run as described above for 22 cycles. PCR products were sent to Kunming Shuoqing Biological Technology Company (China) for Sanger sequencing with an ABI3739XL machine (Applied Biosystems). The 646-bp fragments just between the 3′ ends of the primers were assembled and used for sequence analysis.