Universal dna purification kit

The open reading frame (ORF) of the AfPAL and ArPAL gene from cDNA were amplified based on the annotation of RNA-seq by specific primers (5′-ATGGACCATGCTAGGGAG AACG-3′/5′-CTAGCAAATAGGGAGAGGAGCTTCA-3′) (http://www.premierbiosoft.com/primerdesign/). The amplified fragments were purified using Universal DNA Purification Kit (Tiangen, China), added dATP in the tail of sequences using the TaKaRa TaqTM (TakaRa, China), cloned into pMD19-T vector (TakaRa, China), and sequenced by Shanghai Sangon Biotech Co., Ltd (China).

Denaturing gradient gel electrophoresis (DGGE) of the amplified
16S rDNAgene was performed using an 8% acrylamide gel containing a denaturant gradient of 40-60% (100% defined as 7 M urea and 40% deionized formamide) in 1 × TAE buffer (40 mM Tris, 20 mM acetate, and 1.0 mM Na2-EDTA) at 60°C at a constant voltage of 80 V for 1 hr and 60 V for 16 hr. The gels were stained with ethidium bromide and photographed with UV transillumination. The DNA fragments from the DGGE gels were excised with sterile razor blades immediately after staining and visualization and soaked in sterilized distilled water at 4°C overnight. The supernatant after centrifugation (10,000 ×
g, 5 min at 4°C) was used as the DNA template for the 16S rDNA V3 amplification using the same primers without the GC-clamp. The PCR products were analyzed by electrophoresis on a 1.5% (w/v) agarose gel. The amplification products were purified using a universal DNA purification kit (TIANGEN,
www.tiangen.com) and sequenced.

To detect the expression pattern of five different genes (IDGF1, IDGF3_1, IDGF4_0, IDGF4_1, and IDGF6), total RNA was isolated from eight different developmental stages of Z. cucurbitae. Briefly, A total of ten individuals according to the body size (Per replication: L2 20 larvae, L3-1 10 larvae, L3-2 10 larvae, P-E, P-M, P-L 5 pupae for each, A-E and A-M 2 adults for each) were randomly collected and mixed for RNA extraction. cDNA was synthesized using commercially available HiScript^® III 1st Strand cDNA Synthesis Kit following the manufacturer’s instructions. RT-qPCR was performed to verify IDGFs gene fragment (Supplementary Table 3) from Z. cucurbitae using Prime STAR^® HS DNA Polymerase (Takara, Japan) under the following conditions: initial denaturation at 94°C for 5 min; followed by 30 cycles of Denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C (according to the size of each gene) and final extension at 72°C for 5 min. Amplified products were examined by 1.2% agarose gel electrophoresis and purified using a Universal DNA Purification kit (Tiangen, China). Amplified PCR products were cloned into a pMD^TM18-T vector (Takara, Japan), and verified by sequencing at Sangon Biotech Company Shanghai, China.

Viral RNA/DNA contained in 50 μl of each of 60 serum samples was processed for nucleotide extraction using a MiniBEST Viral RNA/DNA Extraction Kit (Takara, Japan) following the manufacturer's instructions and then diluted in 30 μl of RNA/DNA nuclease-free water. Six microliters of the extracted RNA was then subjected to cDNA synthesis using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) with random primers as the reverse transcription primers. As performed in our previous studies, nested PCR (targeting the EqPV-H VP gene/EqHV NS3 gene) and two rounds of RNA PCR (targeting the EPgV NS3 gene/TDAV NS3 gene) were performed using GenStar Taq Polymerase Premix (Kangrun Chengye, China) with the corresponding PCR procedures and primers.
After PCR, the products were detected by electrophoresis on 1.5% agarose gels. Products with the expected PCR band were considered positive samples. The nucleic acids contained in the agarose gel were then purified using a universal DNA purification kit (Tiangen, China). The purified nucleic acids were sent for direct Sanger sequencing from both ends using the PCR primers for detecting the corresponding viruses (BGI, China). After obtaining the raw sequencing data, the presence of the detected equine viruses (EqPV-H, EqHV, EPgV, and TDAV) was assessed with the online BLAST analysis tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi).