Gelred

To compare the sensitivity of the LAMP developed in this study with the nested PCR (nPCR), which was considered the gold standard for the molecular diagnosis of B. bigemina, the primers, procedures, and programs reported by the World Organization for Animal Health, (23 ) and by Figueroa et al. (24 (link)) were used as references. The analysis of the results was done visually (13 (link)) and on an agarose gel stained with GelRed using a gel documentation equipment (Bio-Rad. Hercules, California, United States).

In this study, nine ingredients that had demonstrated good in vitro biochemical activity in our laboratory, were selected, they have a good ability to scavenge free radicals and inhibit hyaluronidase. They were labeled: oat (Avena sativa) extract (S1), four stamen stephania (Stephania tetrandra) root (S2), stachyose (S3), erythritol (S4), ceramide 3 liposomes (S5), olive (Olea europaea) leaf extract (S6), compound anti-allergic itch-relief remedy (S7/8), and brown algae (Phaeophyceae) extract (S9).
HaCaT (immortalized human epidermal keratinocyte) cells were purchased from the Peking Union Medical College Centre (Beijing, China). DNA markers and real-time PCR components were purchased from Invitrogen Biotechnology (Thermo Fisher Scientific, Waltham, MA, USA). GelRed was obtained from Bio-Rad (Hercules, CA, USA). BD CAB Flex Set was purchased from BD Biosciences (Franklin Lakes, NJ, USA). TRizol was purchased from Ambion (Life Technologies, Carlsbad, CA, USA).
The main instruments employed included: an enzyme labeler (TECAN, Männedorf, Switzerland), a gel imaging analysis system (Alpha Innotech, San Leandro, CA, USA), an Accuri C6 flow cytometer (BD Biosciences), a PCR thermocycler (Eppendorf, Hamburg, Germany), a real-time fluorescence quantitative PCR system (Roche, Basel, Switzerland), and an electrophoresis instrument (Tanon Science & Technology Co., Ltd., Shanghai, China).

Total RNAs were isolated and treated with DNase I on columns using the Qiagen RNeasy Plant Mini Kit (Qiagen, Valencia, CA) following the manufacturer's instructions. Genomic DNA extraction for conventional PCR was done using the QIAGEN DNeasy Plant Mini Kit (Qiagen Sciences Inc, Germantown, MD, USA). For KASP assays, DNA was extracted from 96 plants using the 96-well plate extraction procedure modified from Holleley and Sutcliffe (2022 ). The quality and concentration of total RNA/DNA were assessed using 260/280ABS measurements on a NanoDrop 1,000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA). The integrity of DNA or RNA was checked via agarose gel electrophoresis with 2 μl of a sample, 4 μl of water, 1 μl loading buffer (98% formamide, 10 mM EDTA, 0.25% bromophenol blue, and 0.25% xylene cyanol) on a 0.8–1% gel stained by GelRed (Bio-Rad, Hercules, CA) at 125 volts for 25 min.

To asses recombination in target tissues (GC and oocytes), DNA was extracted and PCR was performed using the following primers: Flox11F (as described above) and Flox14R 5′-GCAGCAGAATACTCTACAGCTC-3′. The Wild type Stat3 allele produces a 2100 bp amplification product with these primers while, if recombination had occurred, a 310 bp product was observed in 2% agarose gel electrophoresis stained with Gel Red (BioRad).

Clonal relationship between representative Salmonella isolates was examined by pulsed-field gel electrophoresis (PFGE) according to the PulseNet PFGE protocol for Salmonella36 (link). S1-PFGE was conducted to determine the size of large plasmids. Briefly, agarose-embedded DNA was digested with S1 nuclease (New England Bio-Lab) at 37 °C for 1 hr. The restriction fragments were separated by electrophoresis in 0.5 Tris-borate-EDTA buffer at 14 °C for 18 h using a Chef Mapper electrophoresis system (Bio-Rad, Hercules, CA) with pulse times of 2.16 to 63.8 S. Phage Lambda PFGE ladder (New England Biolab) was used as DNA size marker. The gels were stained with GelRed, and DNA bands were visualized with UV transillumination (Bio-Rad). Southern blot hybridization was carried out by following the manufacturer’s instructions of the DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics), using the different PMQR gene and bla_CTX-M-64 digoxigenin-labeled probes.