Green taq mix

In order to evaluate the accuracy and effectiveness of the Dot-ELISA and AuNP-ICTS for MCMV detection in actual samples, a total of 20 blinded maize plant samples were analyzed by the two serological assays in this study. At the same time, the monitoring results of the Dot-ELISA and AuNP-ICTS were validated by conventional RT-PCR. For RT-PCR, total RNA was first extracted from each sample (100 mg/each sample) using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and then reverse transcribed with the gene-specific reverse primer, i.e., MCMV-R (5′-ATGGCTCGTGATAAACGGCA-3′), by the HiScript Reverse Transcriptase kit (Vazyme, Nanjing, China). The presence of MCMV in maize samples was then determined using RT-PCR with a set of conserved primers, MCMV-F (5′-GCTCTCGTGAAACACGGACT-3′) and MCMV-R (5′-ATGGCTCGTGATAAACGGCA-3′). Each PCR reaction contained 1 μL of each primer (10 μmol/L), 1 μL of cDNA, 10 μL of 2× Green Taq mix (Vazyme), and 7 μL of sterile deionized water. The reaction cycle was performed as follows: 95 °C for 3 min; 30 cycles of 95 °C for 30 s; 58 °C for 15 s; and 72 °C for 30 s. The final extension was 10 min at 72 °C.

We downloaded the G0S2 full length fragment on NCBI (https://www.ncbi.nlm.nih.gov, accessed on 20 March 2021), and then used NCBI Primer-BLAST (Primer designing tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/)) to design PCR amplification primers. The total volume of PCR was 30 μL, and the amplification system was 3 uL partridge chicken gDNA at a concentration of 100–200 ng/uL; 1.2 μL upstream primer (Tsingke Biotech, Beijing, China) at a concentration of 10nM; 1.2 μL downstream primer at a concentration of 10 nM; 9.6 μL double distilled water; 15 μL Green Taq Mix (Vazyme, Nanjing, China). The primer sequences of PCR were upstream primer 5′-GCTACACTAACGTGCCCCTC-3′, downstream primer 3′-TTACTGCCCACAGGCGTTC-5′. The PCR program was pre-denatured for the first 5 min, denatured at 95 °C for 15 s, followed by annealing at 58 °C, extension at 72 °C for 50 s (32 cycles), and final extension at 72 °C for 5 min at the end of the cycle. It was stored temporarily at 4 °C until off the machine. Then, the amplified samples were sent to Tsingke Biotech Co., Ltd. for sequencing.

Five housekeeping genes (ugpB, pilT, lepA, trpB, and gltA) were amplified by PCR using primer pairs reported by Feng et al. (2009) (link). The 50 μL reaction mix included 1.0 μL bacterial genomic DNA (100 ng/μL), 20 μL Green Taq Mix (Vazyme Biotech, Nanjing, China), 1.0 μL each of forward and reverse primers (10 μmol/L), and 27 μL sterile water. The PCR program was 5 min at 95°C for initial denaturation; 35 cycles of 30 s at 95°C for denaturation, 30 s at 60°C for annealing, and 30 s at 72°C for extension; and 5 min at 72°C for final extension. The expected amplification product had a size of 444, 398, 489, 434, and 481 bp for ugpB, pilT, lepA, trpB, and gltA, respectively. Bacterial genomic DNA of Aaa strain ATCC 19860 (Institute of Microbiology, Chinese Academy of Sciences) was used as the positive control. Sterile distilled water was used as the blank control. All primer information is given in Table S2.

Total RNA was purified using the Total RNA Miniprep Kit (#AP-MN-MS-RNA, Axygen, Union City, CA, USA). Total RNA was treated with DNase I (#M1682, Thermo Scientific, Carlsbad, CA, USA) to remove genomic DNA contamination. Then, reverse transcription was performed using the Maxima H Minus cDNA Synthesis Master Mix (#M1682, Thermo Scientific, Carlsbad, CA, USA) by following the manufacturer’s protocol. Semiquantitative PCR was performed using Green Taq Mix (#P131, Vazyme Biotech, Nanjing, China). Quantitative real-time PCR was performed using Universal SYBR Green Fast qPCR Mix (#RK21203, Abclonal, Wuhan, China). The primers involved in these experiments are listed in Table S2.

Bacterial cells obtained through functional selection were utilized in colony PCR reactions to amplify the inserts. The PCR reactions employed the M13 primer pair, with 4 μL of 10 μM primers, 25 μL of Green Taq Mix (Vazyme biotech, P131), 4 μL of bacterial suspension, and 17 μL of ddH2O, resulting in a final volume of 50 μL. DNA amplification was conducted in a thermocycler at 95°C for 10 min, followed by 30 cycles of 95°C for 15 s, 55°C for 15 s, and 72°C for 4 min. The final extension was conducted at 72°C for 7 min. Gel recovery of PCR samples was carried out using the Gel Mini Purification Kit (Zomanbio, ZP202), adhering to the manufacturer’s instructions.

Genomic DNA was isolated from the candidate rTTT0 plants using the Plant Genomic DNA Kit (TIANGEN). The cell type identification of rTTT0 plants was performed according to Li et al. with minor modifications [17 (link)]. Briefly, each 20-μl reaction system contained 50 ng of genomic DNA, 500 nM specific primers (atpA, listed in Supplementary Data Table S3), and 2 × Green Taq Mix (Vazyme). PCR was performed using the following program: 95°C for 5 minutes followed by 30 cycles of 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 15 seconds, then 72°C for 5 minutes. The genomic DNA of tuber mustard and red cabbage was amplified into 1050- and 1500-bp fragments, respectively.