SN/T 5198-2020Technical specifications for the quarantine of Bumblebee Brachyhymenium (English PDF)

4.1 Equipment

4.1.1 Autoclave.

4.1.2 Gradient PCR amplification instrument.

4.1.3 Fluorescent PCR machine.

4.1.4 High-speed refrigerated centrifuge (rotating speed 12 000 r/min).

4.1.5 Micro pipette.

4.1.6 Biological safety cabinet.

4.1.7 DNA relative molecular quality standards (100 bp ~1 000 bp).

4.1.8 Horizontal electrophoresis apparatus.

4.1.9 Gel imaging system.

4.1.10 Electronic balance (with a sense of 0.01 g).

4.1.11 Refrigerator (2 °C~8 °C and -20 °C).

4.2 Reagents

4.2.1 Water. Meet the first grade water specification in GB/T 6682.

4.2.2 Liquid nitrogen.

4.2.3 Anhydrous ether.

4.2.4 Absolute ethanol.

4.2.5 0.01 mol/L phosphate (PBS) buffer, the preparation method is shown in Appendix A, A.1.

4.2.6 10% SDS solution, the preparation method is shown in Appendix A, A.2.

4.2.7 Proteinase K.

4.2.8 5 mol/L NaCl solution, the preparation method is shown in Appendix A, A.3.

4.2.9 CTAB/NaCl solution, the preparation method is shown in Appendix A, A.4.

4.2.10 Trichloromethane/isoamyl alcohol mixture, the preparation method is shown in Appendix A, A.5.

4.2.11 Phenol/chloroform/isoamyl alcohol mixture, the preparation method is shown in Appendix A, A.6.

4.2.12 Isopropanol.

4.2.13 TE buffer.

4.2.14 10×PCR buffer.

4.2.15 dNTPs (including dCTP, dGTP, dATP, dTTP).

4.2.16 Taq DNA polymerase.

4.2.17 Agarose.

4.2.18 0.5×TBE buffer, the preparation method is shown in Appendix A, A.7.

4.2.19 Loading buffer, the preparation method is shown in Appendix A, A.8.

4.2.20 EB (10 mg/mL). For the preparation method, see A.9 in Appendix A.

4.2.21 DNA relative molecular mass standard Marker.

4.2.22 DNA purification and recovery kit.

4.3 PCR primers

Designed according to the nucleic acid sequence of the ITS gene of Bumblebee Bremenworm, the amplified length is 447 bp (see Appendix B in B.1), and it is dissolved in water to 10 After µmol/L, store at -20 °C for later use.

4.4 Real-time PCR primers and probes

4.5 Quality control substances

Use the bumblebee's abdominal tissue or DNA containing the target fragment infected with Bombus brachymemma as a positive control, and use the uninfected Bombus brachymembranaceus the healthy bumblebee abdomen tissue of the insect was used as a negative control, and water was used as a blank control.

5.1 Sampling ratio

5.1.1 When the number of bee colonies is less than or equal to 10 colonies, all bee colonies are sampled.

5.1.2 When the number is between 11 and 100 groups, based on the sampling volume of 10 groups, each additional 10 groups will increase the sampling volume by 1 group.

5.1.3 When the number of bees is from 101 to.200 groups, 15% of the total number of bee groups shall be sampled.

5.1.4 When the number of bee colonies is greater than or equal to.201 colonies, sampling is based on 10% of the total colony numbers.

5.2 Sampling method

Take 10 to 20 bumblebees with weaker vigor from the bee colony to be inspected, put them into the beaker and cover it, and record the group number. Use live bees before quarantine Put them to death or place them in the freezer of the refrigerator for 10 min.

6.1 DNA extraction

6.1.1 Cut the bumblebee abdomen tissue sample and place it in a sterilized mortar, add an appropriate amount of liquid nitrogen and repeatedly grind it into a powder.

Grind into a homogenate with phosphate buffer, transfer to a 1.5 mL centrifuge tube, centrifuge at 12,000 r/min for 2 min, discard the supernatant, and add 50 µL TE buffer, after fully suspending the sample, add 60 µL of 10% SDS solution and 10 µL of 20 mg/mL proteinase K, and mix well at 37 °C Incubate for 1 h.

6.1.2 Add 100 µL of 5 mol/L NaCl solution, turn upside down and mix thoroughly, add 80 µL of CTAB/NaCl solution, and mix well at 65 °C Incubate for 10 min; add 700 µL of chloroform/isoamyl alcohol mixture (volume ratio 24.1), mix, and centrifuge at 12,000 r/min for 5 min.

6.1.3 Pipette the supernatant to another clean centrifuge tube, add an equal volume of phenol/chloroform/isoamyl alcohol mixture (the volume ratio of the three is 25.24.1), mix upside down, and centrifuge at 12,000 r/min for 5 min.

6.1.4 Pipette the supernatant to another clean centrifuge tube, add 2 times the volume of pre-chilled isopropanol to precipitate the DNA, mix gently, and place at 4 °C Centrifuge at 12 000 r/min for 15 min to completely remove the supernatant.

6.1.5 Wash the precipitate with 600 µL of 70% pre-cooled ethanol, centrifuge at 12 000 r/min for 10 min at 4 °C, discard the supernatant, and then centrifuge briefly, and use a pipette.

The liquid dispenser completely absorbs the alcohol, and it is allowed to dry naturally on the ultra-clean workbench.

6.1.6 Add 20 µL of TE solution to dissolve DNA and store at -20°C. Equivalent commercial kits can also be used for DNA extraction.

6.2 PCR amplification

6.2.1 Reaction system

The general PCR reaction system is shown in Table 1.For PCR amplification, an equivalent commercial DNA amplification kit can be used.

Note. The amount of each reagent in the reaction system can be adjusted appropriately according to the total volume of the reaction system.

6.2.2 Reaction procedure

Pre-denaturation at 94 °C for 5 min; afterwards, denaturation at 94 °C for 45 s, annealing at 59 °C for 30 s, extension at 72 °C for 45 s, a total of 35 cycles; 72 °C supplementation Extend for 10 min and keep at 4 °C. At the same time, a positive control, a negative control and a blank control are set up.

6.3 Agarose gel electrophoresis

Prepare a 1.5% agarose gel with TBE running buffer. Put the gel into the horizontal electrophoresis tank so that the electrophoresis buffer just floods the gel surface. Mix 6 µL of sample and 2 µL of loading buffer in proportion and add to the sample well. Use DNA relative molecular mass standards during electrophoresis for comparison. 5 V/cm electrophoresis is about 0.5 h, and stops when the indicator reaches the bottom.

6.4 Gel imaging analysis and sequencing

Put the electrophoresis gel into a solution containing 0.5 µg/mL EB and stain for 10 min to 30 min (or equivalent commercial nucleic acid dye Dissolve directly in the gel when placing the agarose gel), rinse the gel in water, observe and judge the result with a gel imager,...