GB/T 36875-2018Method of RT-nPCR for detection of classical swine fever virus (English PDF)

3.1 Laboratory conditions

3.1.1 Laboratory partition

The PCR laboratory partition includes a nucleic acid extraction region, a PCR reaction system preparation region---dispensing region, an amplification region, and an electrophoresis region. The sequence of processes is Nucleic acid extraction zone->displacing zone->amplification zone->electrophoresis zone. Dedicated to instruments and consumables in each area.

3.1.2 Instruments and equipment to be equipped in the laboratory

3.1.2.1 Instruments. Class II biosafety cabinet, desktop high speed refrigerated centrifuge, ordinary refrigerator (-20 ° C and 4 ° C), analytical balance, PCR amplification Instrument, electrophoresis, electrophoresis tank, UV gel imager (or UV analyzer), tissue grinder, tonsil sampler, microwave oven, single channel transfer Liquid filter (2 µL, 10 µL, 200 µL, 1000 µL).

3.1.2.2 Equipment. sterile syringe, ophthalmic scissors, ophthalmology, weighing paper, cotton swabs, sterilized toothpicks, 500mL measuring cylinders, 500mL conical flasks, One-time RNase-free tips (10 µL, 200 µL, 1000 µL), 1.5 mL RNase-free tubes, and 0.2 mL thin-wall PCR tubes.

3.2 Chemicals and reagents

3.2.1 Total RNA extraction reagent Trizol

3.2.2 Preparation of TAE electrophoresis buffer, see Appendix A for preparation methods.

3.2.3 1% agarose gel, see Appendix A for the preparation method.

3.2.4 Other reagents. 200U/µLM-MLV reverse transcriptase, 5×M-MLV buffer, 40U/µL RNase inhibitor, DEPC- H2O, 5U/µLExTaq DNA polymerase, 10×ExTaq buffer (Mg2 Plus), 6 base random primer (50 µmol/L), 6× loading buffer, dNTPs (2.5mmol/L/dNTP), isopropanol, chloroform, 75% ethanol, ddH2O, ethidium bromide instead […]