SN/T 5283-2020Technical Specification for Bumblebee Nosema quarantine (English PDF)

4.1 Masher

4.2 Optical phase contrast microscope

4.3 Autoclave

4.4 Thermal cycler

4.5 Fluorescence PCR machine

4.6 High-speed refrigerated centrifuge

4.7 Micropipette (0.5 µL~10 µL, 2 µL~20 µL, 20 µL~200 µL, 100 µL~1000 µL)

4.8 Clean bench

4.9 Horizontal Electrophoresis Apparatus

4.10 Gel imaging system

4.11 Electronic balance (sensitivity 0.01 g)

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

5.13 Common PCR primers

5.14 Fluorescent PCR primers and probes

6.1 Sampling ratio

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

6.1.2 When the number is between 11 clusters and 100 clusters, based on the sampling volume of 10 clusters, each additional 10 clusters will increase the sampling volume by 1 cluster.

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

6.1.4 When the number of bee colonies >.200 colonies, based on the sampling amount of.200 colonies, each additional 20 colonies will increase the sampling quantity by 1 colony.

6.2 Sampling method

Take 30 bumblebees with weaker vigor from each selected bee colony, put them into the beaker and cover it, and record the colony number. Use live bees before inspection Put them to death or place them in the freezer of the refrigerator for 10 min.

7.1 Morphological detection method

7.1.1 Sample processing

For bee samples after sacrifice or fresh bee samples after death, take the intestines, add physiological saline according to 1.10, and fill with a masher Divide and mash (for dried dead bee samples, first cut off the head and chest, take the abdomen, soak for 12 hours and then mash). Yarn for homogenate Filter through a net (50 mesh to 100 mesh) and collect the filtrate. Differential centrifugation of the filtrate (500 r/min, centrifugation for 1 min, discard the precipitate, and then supernatant Centrifuge at 5 000 r/min for 10 min, discard the supernatant) 2 to 3 times. Suspend the sample with 1 to 10 times the volume of normal saline, as Microsporidian liquid to be tested.

7.1.2 Microscopic examination

Take out 50 µL of 2 test solution samples from the test solution prepared in 7.1.1, drop them on a glass slide, cover with a cover glass, and use 40×15 (or

10) Double optical phase-contrast microscopes are checked by 2 people, first look at the middle, adjust the fine-tuning knob, and slowly move around to ensure that there are many samples for each in 20 fields of view.

7.1.3 Judgment of results

Under the optical phase contrast microscope, it can be seen that the length is about 5 µm~7 µm and the width is about 3 µm~4 µm. The external shape is short and wide, with blue refraction.

Oval particles with dark edges are microsporidia and are judged as positive (see Appendix B).

7.2 PCR detection method

7.2.1 Primer

Designed according to the nucleic acid sequence of the 16S rRNA gene of Nosema bombycis, the amplified length is about 213 bp, and it is dissolved with ddH2O to After 10 µmol/L, store at -20 °C for later use.

7.2.2 Setting up a comparison

Blank control, negative control and positive control should be set for each reaction. The blank control can replace the sample with ddH2O; the negative control is Abdominal tissues of healthy bumblebees that were not infected with Nosema bombyma; the positive control was the abdomen tissues of bumblebees infected with Nosema bomby.

7.2.3 DNA extraction

7.2.3.1 Take 0.5 mL of the 7.1.1 test solution and transfer to a 1.5 mL centrifuge tube, add 0.5 mL of 0.2 mol/L KOH, and fully Mix, induce at 27 °C for 1 h, centrifuge at 5,000 r/min for 5 min, and discard the supernatant.

7.2.3.2 Resuspend with 0.5 mL of normal saline and place at 25°C for 30 min. Add 0.5 mL saturated phenol to each tube and mix thoroughly for 5 min by inverting.

Centrifuge for 10 min at 12 000 r/min at 4 °C.

7.2.3.3 Take the water phase to another clean centrifuge tube, add an equal volume of phenol/chloroform/isoamyl alcohol (the volume ratio of the three is 25.24.1), mix upside down for 5 min, and centrifuge at 12 000 r/min at 4°C for 10 min.

7.2.3.4 Take the water phase to another clean centrifuge tube, add an equal volume of chloroform/isoamyl alcohol (the volume ratio of the three is 24.1), up and down Mix by inversion for 5 min, and centrifuge at 12 000 r/min at 4 °C for 10 min.

7.2.3.5 Take the water phase to another clean centrifuge tube, add 2 times the volume of pre-cooled isopropanol to precipitate DNA, mix gently, 4 °C 12 000 r/min Centrifuge for 15 min, and completely remove the supernatant.

7.2.3.6 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 centrifuge again.

Use a pipette to thoroughly remove the alcohol, and let it dry naturally on a clean bench.

7.2.3.7 Add 20 µL of TE solution to dissolve DNA and store at -20°C.

7.2.3.8 Blank control, negative control and positive control start synchronous operation from 7.1.1.

7.2.3.9 DNA extraction can also choose equivalent commercial kits.

7.2.4 PCR amplification

7.2.4.1 Reaction system

The general PCR reaction system is shown in Table 1.PCR amplification can also use equivalent commercial DNA amplification kits.

7.2.4.2 Reaction procedure