Rna rapid concentration and purification kit

Salmonella-specific invA gene was retrieved from GenBank, and the conservative sequence region was selected to design RPA amplification primers using Primer 5 software, and Primer-BLAST software was used to verify the specificity of the primer sequence. The designed primers, probes, and oligonucleotides were synthesized by Sangon Biotech (Shanghai, China). The oligonucleotides containing T7 promoter, repeat, and spacer sequences were annealed with a T7 primer. Then, the crRNA was synthesized by incubating at 42°C for 2 h with T7 RNA polymerase (TaKaRa, China). The synthesized crRNA was digested with DNase I (TaKaRa, China) at 37°C for 1 h followed by purification with RNA rapid concentration and purification kit (Sangon Biotech, China) according to the manufacturer’s protocol. The concentration of crRNA was quantified using Qubit 2.0 (Invitrogen, United States). All nucleic acid sequences used in this study are shown in Table 1.

The templates for an oligonucleotide incorporating T7 promoters, repetitive sequences, and interval sequences have been developed to create the double strand DNA (dsDNA). Forward and reverse oligonucleotide DNA (5 μmol/L) were added to produce dsDNA templates for transcription. And each single-chain template was then mixed, annealed for 5 min at 95°C and naturally cooled to room temperature naturally. The crRNA was synthesized by incubating the above mixtures for 2 h at 42°C with T7 RNA polymerase (TaKaRa). To carry out the process, the synthesized crRNA was digested with DNase I (TaKaRa) at 37°C for 1 h to remove DNA templates, and then purified using the RNA Rapid Concentration and Purification kit (Sangon Biotech, Shanghai, China) in accordance with the manufacturer’s instructions. The crRNA concentration was then determined using the NanoDrop spectrophotometer (Thermo Fisher Scientific, Shanghai, China) and was subsequently kept at-80°C until use.