Dl2000 dna ladder

Genotyping of E. coli was performed using a triplex PCR assay with a combination of three gene markers (ChuA, yjaA, and tspE4C2) (27 (link), 37 (link)). The resulting PCR products allowed classification of the strains into one of the four major phylogenetic lineages: A1, B1, B2, and D. The primer sequences for the PCR and the sizes of the amplified products are listed in Table 2. The primers were synthesized by Sangon Biological Engineering Technology and Service Co., Ltd. (Shanghai, China).
Each PCR was performed in a final volume of 25 µL, which included 12.5 µL of 2× PCR Mix (TransGen, Beijing, China), 0.5 µL of 10 nmoL of each primer, 11 µL of ddH₂O, and 1 µL of bacterial culture. All PCRs were performed with an initial denaturation step at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and a final single extension step at 72°C for 5 min. PCR products were detected with electrophoresis in 1% agarose gels, along with a DL2000 DNA ladder (TaKaRa, Dalian, China), and visualized under UV illumination after staining with ethidium bromide.

To identify the causative agent of the disease, the potential viral and bacterial pathogens were examined. The bacteriological culture was performed as described previously (26 (link)). The samples from the same duckling were combined, and processed by extracting RNA using the EasyPure Viral RNA Kit (Takara, Shanghai). Subsequently, the RNA samples were used to detect MDRV, DHAV-1, DHAV-3, NDV, DTMUV, and NDRV using RT-PCR protocols as described previously (Supplementary Table S1) (27 (link)). The PCR products were collected by electrophoresis in 1.0% agarose gel with DL2000 DNA Ladder (Takara, Shanghai).