SN/T 5228.1-2019Rapid screening method for pathogenic microorganisms in exported foods MALDI-TOF MS method Part 1: Vibrio alginolyticus (English PDF)

Foreword

SN/T 5228-2019 "The MALDI-TOF MS Method for Rapid Screening of Pathogenic Microorganisms in Exported Foods" is divided into 9 parts.

--Part 1: Vibrio alginolyticus;

--Part 2: Clostridium perfringens;

--Part 3: Staphylococcus aureus;

--Part 4: Cronobacterium;

--Part 5: Vibrio vulnificus;

--Part 6: Bacillus cereus;

--Part 7: Campylobacter jejuni;

--Part 8: Klebsiella pneumoniae;

--Part 9: Pseudomonas aeruginosa.

This part is part 1 of SN/T 5228-2019.

This part was drafted in accordance with the rules given in GB/T 1.1-2009.

This part is proposed and managed by the General Administration of Customs of the People's Republic of China.

Drafting organization of this section. Beijing Customs of the People's Republic of China

1 Scope

This part of SN/T 5228 specifies the MALDI-TOF MS rapid screening method for Vibrio alginolyticus in export food.

This section applies to the rapid screening of Vibrio alginolyticus in export food.

2 Normative references

The following documents are indispensable for the application of this document. For dated reference documents, only the dated version applies to this article Pieces. For undated reference documents, the latest version (including all amendments) is applicable to this document.

GB/T 6682 Analytical laboratory water specifications and test methods GB 19489 Laboratory Biosafety General Requirements Bacteriological Analysis Handbook Chapter 9 Vibrio Test (US FDA, Eighth Edition)

3 Abbreviations

The following abbreviations apply to this document.

4 Biosecurity measures

In order to protect the safety of laboratory personnel, qualified personnel should detect Vibrio alginolyticus, and all cultures and wastes should be refer to the relevant regulations in GB 19489.

5 Method summary

MALDI-TOF MS is a technology applied to the rapid detection of microbial whole cells, mainly based on the fingerprint analysis of microbial characteristic protein.

Complete the identification and classification of microorganisms. Place the sample of the colony to be identified and an appropriate amount of matrix solution on the sample plate, and form after the solvent volatilizes the co-crystallization of the sample and the matrix, the laser is used as the energy source to radiate the co-crystal, the matrix molecules absorb energy and desorb the sample and make the sample Product ionization, after the time-of-flight analyzer, separates the ions of different mass-to-charge ratios to form a microbial-specific mass spectrum. Will be tested Comparing the substance spectrum with the standard protein fingerprint database of known microorganisms, the species of microorganisms can be determined, and the species of microorganisms can be determined.

是identification.

6 Reagents and materials

Except for special instructions, all reagents are chromatographically pure reagents. The experimental water meets the requirements of first-grade water in GB/T 6682.

6.1 Blood agar plate.

6.2 Acetonitrile (acetonitrile, ACN).

6.3 Absolute ethanol.

6.4 Formic acid.

6.5 Trifluoroacetic acid (TFA).

6.6 alpha-cyano-4-hydroxycinnamic acid (CHCA).

6.7 BTS standard product.

6.8 Solvent I. According to the ratio of water. acetonitrile (6.2). trifluoroacetic acid (6.5) to 50.47.5.2.5 (volume ratio), now Equipped for immediate use.

6.9 Preparation of BTS standard solution. Dissolve BTS standard (6.7) with 50 µL solvent I (6.8). Pipette repeatedly to dissolve BTS powder (avoid vigorous shaking), leave it at room temperature for 5 minutes, and dissolve again. After the BTS standard (6.7) is completely dissolved, centrifuge it immediately.

Use.200 µL PCR tubes for aliquoting, 5 µL per tube, and store at -20°C for later use.

6.10 Preparation of CHCA matrix solution. Add solvent I (6.8) to CHCA (6.6) powder to make the final concentration of CHCA 10 mg/mL.

Shake and mix until the solution is clear. Use 1.5 mL centrifuge tubes for aliquots, 50 µL per tube, seal the mouth of the tube with a parafilm, and store at room temperature for later use.

After storage, if a large amount of precipitation occurs, it should be discarded.

7 Main instruments and equipment

7.1 Bench-top centrifuge. the maximum centrifugal force is >=16 000 g.

7.2 Vortex oscillator.

7.3 Micro-adjustable pipettes and sterile tips. 2 µL, 100 µL,.200 µL, 1000 µL.

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

8 Detection steps

9 Results judgment and report

9.1 The suspicious colony is identified as Vibrio alginolyticus by MALDI-TOF MS, and the matching score is greater than or equal to 1.700, the test result can be determined to be positive If the sex is suspicious, it should be further confirmed and reported in accordance with the "Bacteriological Analysis Manual Chapter 9 Vibrio Test".

9.2 For results other than 9.1, the test result can be judged to be negative, and it is reported that no Vibrio alginolyticus has been detected.

10 Waste treatment and pollution prevention measures

The waste in the testing process needs to be autoclaved at 121°C for at least 30 minutes before being disposed of.