SN/T 5204-2020Plant pathogenic bacteria screening MALDI-TOF MS method (English PDF)

5.1 Apparatus

Autoclave, light incubator, balance (sensitivity 1/100; 1/10000), water bath, pure water meter, vortex oscillator, desktop Centrifuge, refrigerator, ultra-clean workbench, matrix-assisted laser desorption ionization time-of-flight mass spectrometer, ultrasonic vibrator.

5.2 Utensils

Petri dishes, graduated cylinders, pipettes, alcohol lamps, filter paper, micropipettes (0.5 µL, 2 µL, 10 µL, 20 µL, 100 µL,.200 µL, 1 000 µL), PCR reaction tube (0.2 mL, 0.5 mL, 1 mL), Tip head (0.1 µL~10 µL, 5 µL~200 µL, 100 µL~1000 µL), Mass spectrometry analysis of the sample target.

7.1 Strain culture

The test strains were streaked inoculated into NBY and other related media for activation and culture, and then incubated in the dark at 28 °C ± 2 °C for 48 h before use.

7.2 Bacteria treatment

See Appendix B for specific steps.

7.3 Mass spectrometry analysis

7.3.1 Instrument conditions

7.3.1.1 Instrument. Matrix-assisted laser desorption ionization time-of-flight mass spectrometer.

7.3.1.2 Laser energy. According to the sample, it can be adjusted from 0% to 100%.

7.3.1.3 Mass spectrum scanning range. 1000 Da~20000 Da.

7.3.1.4 Detection method. linear positive ion detection method.

7.3.1.5 Microbial fingerprint library. the instrument's own database or self-built equivalent type database.

7.3.2 Calibration of quality control standards

It is recommended to use a mixture of Peptide Calibration Standard and Protein Calibration Standard I before testing each sample.

The mass spectrometer is calibrated with the characteristic peaks of liquid or equivalent standards.

7.3.3 Spot

7.3.3.1 Spot 1 µL of supernatant on the mass spectrometer target, 4 replicates for each sample spot.

7.3.3.2 Take 1 µL of standard calibration solution as a control.

7.3.3.3 After natural drying at room temperature, spot 1 µL of substrate as soon as possible and let it dry.

7.3.4 Identification

Put the sample target board into the instrument, set parameters such as laser energy and mass spectrum scan range, and collect mass spectrum data. And then by the processed mass spectrum data is automatically/manually imported into the local microbial spectrum library, and the obtained mass spectrum analysis spectrum and microbial fingerprint spectrum library are imported into the library.

Line comparison, the log (score) value is calculated through the instrument's own software, and the type of unknown bacteria is judged.

9.1 Sample storage

The stored samples should be stored according to the state of the samples and should be properly stored for 6 months for re-inspection, if trade disputes are involved it should be saved until the dispute is resolved.

9.2 Save the original data

After the sample is tested, the original record sheet, spectrum and inspection report or certificate shall be filed and kept in a safe place for re-inspection, negotiation and arbitration.

9.3 Strain preservation

The isolated strains are stored in ultra-low temperature or freeze-dried for re-inspection, negotiation and arbitration.

Appendix A

(Normative appendix) Reagents, solutions and media A.1 Reagents Anhydrous ethanol, isopropanol, acetonitrile, trifluoroacetic acid (TFA) and methanol, the above organic solvents are all chromatographic grade; sterile deionized Water, Alpha-Cyano-4-hydroxycinnamic acid (HCCA), Peptide Calibration Standard and Protein Calibration Standard I.

A.2 Solution

A.2.1 Matrix solution. weigh 5 mg HCCA and dissolve in 500 µL acetonitrile. 10% trifluoroacetic acid. sterile deionized water (2.1.1) in the preparation, a matrix solution that forms a saturated solution of about 10 mg/mL is ready for use. Prepare fresh before each use.

A.2.2 Standard calibration solution. select 1 tube of Peptide Calibration Standard and Protein Calibration Standard I respectively, add Add 125 µL of 0.1% TFA 50% acetonitrile solvent, mix thoroughly, and store at -20 °C for later use. Or use the same type of standard solution.

A.3 Medium

A.3.1 Nutrient Broth Yeast Medium (NBY). beef extract 3 g, peptone 5 g, yeast powder 2 g, dipotassium hydrogen phosphate trihydrate 2.6 g, 0.5 g of potassium dihydrogen phosphate, 2.5 g of glucose, 1000 mL of distilled water, sterilized at 121 °C for 15 min, then add 1 mol/L magnesium sulfate 1 mL of liquid; for NBY solid medium, 15 g of agar powder needs to be added.

A.3.2 Nutrient peptone culture medium (NB). beef extract 3 g, peptone 5 g, distilled water 1000 mL, pH=7.0~7.2, 121 °C Sterilize for 15 min. If solid medium (NA) is required, add 15 g of agar powder.

Appendix B

(Normative appendix) Bacterial treatment B.1 Washing Use a sterile inoculation needle or pipette tip to pick the colonies and weigh 1 mg~2 mg 1.5 mL with 1000 µL sterile deionized water and centrifuge in the tube, shake and mix well, centrifuge at 10 000 r/min for 2 min, and discard the supernatant.

B.2 Solvent treatment Add 300 µL of sterilized deionized water and shake to mix well, add 900 µL of absolute ethanol and shake to mix well, centrifuge at 10 000 r/min 2 min, discard the supernatant and leave the pelleted cells for later use (if necessary, centrifuge again to remove ethanol and water as much as possible).

B.3 Protein precipitation After adding.200 µL of 0.1% TFA, centrifuge, discard the supernatant, add 25 µL of 70% formic acid, mix carefully, and then add 25 µL Acetonitrile, mix well, sonicate for 10 min under temperature-controlled ice bag, centrifuge at 10 000 r/min for 2 min, take the supernatant for quality Spectrum Analysis.