ICS 65.150 B 50 DB37 DB37/T 4058 2020 Inspection protocol for bacterial pathogens of aquatic animals 2020-07-09 2020-08-09 DB37/T 4058 2020 I GB/T 1.1 2009 TC03 DB37/T 4058 2020 1 1 Bacterial pathogens of aquatic animals 2 GB 4789.28 SC/T 7014 2006 SC/T 7201.1 1 SN/T 2123 3 GB 4789.28 A 4 SC/T 7201.1 5 5.1 5.2 SC/T 7014 2006 6 5.3 SN/T 2123 6 6.1 DB37/T 4058 2020 2 6.1.1 28 30 24 h 15 18 24 h 6.1.2 30 g/1 000 mL 28 30 24 h 15 18 24 h 6.2 6.2.1 6.1.1 6.2.2 6.1.2 6.3 6.2 7 7.1 16S rDNA 7.1.1 PCR B 7.1.2 7.1.2.1 PCR DNA Marker 1472 bp DNA PCR 7.1.2.2 DNA Marker 7.1.2.3 DNA Marker DNA 1472 bp DNA PCR 7.1.3 DB37/T 4058 2020 3 7.1.2 NCBI 7.2 SC/T 7014 2006 9.4 7.3 7.1.3 7.2 DB37/T 4058 2020 4 A A A A.1 TE A.1.1 A 1 mol/L Tris HCl(pH8.0)Tris 6.06 g 40 mL HCl 2 mL pH 8.0 50 mL A.1.2 B 0.5 mol/L EDTA(pH8.0)Na2EDTA 2H2O 9.31 g 35 mL 1 g NaOH pH 8.0 50 mL Na2EDTA 2H2O NaOH pH 8.0 A.1.3 TE A 5 mL B 1 mL 400 mL pH 8.0 500 mL A.2 1 TAE Tris 242.0 g Na2EDTA 2H2O 37.2 g 1 L 800 mL 57.1 mL 1 000 mL 50 TAE 50 DB37/T 4058 2020 5 B B B PCR B.1 DNA 0.2 mL PCR 10 L 1 TE A.1 100 5 min-20 5 min 2 5 000 r/min 1 min DNA PCR DNA B.2 PCR 50 L PCR DNA 0.2 ng/L 2 ng/L DNA 10 PCR Buffer Mg2+1.5 mM 5 L dNTP 10 mM 1 L 27F 5 AGA GTT TGA TCC TGG CTC AG 3 1492R 5 TAC GGC TAC CTT GTT ACG ACT T 3 25 M 2 L Taq 2 U/L 1 L 2 L 50 L PCR 95 5 min 30 94 30 s 50 30 s 72 30 s 72 10 min 4 PCR B.3 DNA DNA DNA DNA 1 TAE A.2 2%PCR 6 DL2000 DNA Marker 10 V/cm 30 min _