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SN/T 5228.6-2019 PDF English

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SN/T 5228.6-2019: (Rapid screening method for pathogenic microorganisms in exported foods MALDI-TOF MS method Part 6: Bacillus cereus)
Status: Valid

Std ID	Contents [version]	USD	STEP2	[PDF] delivered in	Standard Title (Description)	Status
SN/T 5228.6-2019	English	169	Add to Cart	3 days [Need to translate]	(Rapid screening method for pathogenic microorganisms in exported foods MALDI-TOF MS method Part 6: Bacillus cereus)	Valid

Standard similar to SN/T 5228.6-2019

GB 5413.12 | GB 5413.10 | SN/T 1888.4 | SN/T 5228.2 | SN/T 5228.3 | SN/T 5228.1 |

Basic data

Standard ID	SN/T 5228.6-2019 (SN/T5228.6-2019)
Description (Translated English)	(Rapid screening method for pathogenic microorganisms in exported foods MALDI-TOF MS method Part 6: Bacillus cereus)
Sector / Industry	Commodity Inspection Standard (Recommended)
Classification of Chinese Standard	C53
Classification of International Standard	67.050
Word Count Estimation	7,787
Date of Issue	2019
Date of Implementation	2020-07-01
Issuing agency(ies)	General Administration of Customs

SN/T 5228.6-2019: (Rapid screening method for pathogenic microorganisms in exported foods MALDI-TOF MS method Part 6: Bacillus cereus)

---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
Rapid detection of pathogens in export food-MALDI-TOF MS method- Part 6.Bacillus cereus The People's Republic of China Entry-Exit Inspection and Quarantine Industry Standards 2019-12-27 release 2020-07-01 Implementation Issued by the General Administration of Customs of the People's Republic of China

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 6 of SN/T 5228-2019. This part was drafted in accordance with the rules given in GB/T 1.1-2009. This standard was proposed and managed by the General Administration of Customs of the People's Republic of China. Drafting organizations of this section. Chinese Academy of Inspection and Quarantine, Jilin Customs of the People’s Republic of China, Yining of the People’s Republic of China Customs, Beijing Customs of the People's Republic of China. The main drafters of this section. Chen Ying, Wang Ping, Zhao Xiaojin, Cai Yang, Meng Ru, Zeng Jing. MALDI-TOF MS method for rapid screening of pathogenic microorganisms in exported food Part 6.Bacillus cereus

1 Scope

This part of SN/T 5228 specifies the MALDI-TOF MS rapid screening method for Bacillus cereus in exported food. This section applies to the rapid screening of Bacillus cereus 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 4789.14 National Food Safety Standard Food Microbiological Inspection Inspection of Bacillus cereus GB/T 6682 Analytical laboratory water specifications and test methods GB 19489 Laboratory Biosafety General Requirements

3 Abbreviations

The following abbreviations apply to this document. BTS. bacterial test standard. CHCA. α-cyano-4-hydroxy-cinnamic acid (α-cyano-4-hydroxy-cinnamic acid). MALDI-TOF MS. matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry).

4 Biosecurity measures

In order to protect the safety of laboratory personnel, qualified personnel should detect Bacillus cereus, all cultures and waste Objects should be implemented with reference 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 Brain Heart Infusion Agar (BHIA). 6.2 Acetonitrile (acetonitrile, ACN). 6.3 Absolute ethanol. 6.4 Trifluoroacetic acid (TFA). 6.5 α-cyano-4-hydroxycinnamic acid (CHCA). 6.6 Formic acid. 6.7 BTS standard product. 6.8 Solvent I. prepared according to the ratio of water. acetonitrile (6.2). trifluoroacetic acid (6.5) to 50.47.5.2.5 (volume ratio). Now 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 then dissolve it again. After BTS (6.7) is completely dissolved, centrifuge it immediately and use 200 µL PCR tubes are aliquoted, 5 µL per tube, stored 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 Micropipettes. 10 µL, 100 µL,.200 µL, 1000 µL. 7.4 Matrix-assisted laser desorption ionization time-of-flight mass spectrometer.

8 detection steps

8.1 Detection flow chart The MALDI-TOF MS detection process of Bacillus cereus in food is shown in Figure 1. 8.2 Sample preparation, enrichment culture and separation The preparation, enrichment culture and separation steps of Bacillus cereus test samples in food are carried out in accordance with the method of GB 4789.14. 8.3 Treatment of suspicious colonies 8.3.1 Inoculation and culture At least 5 suspicious colonies should be picked, streaked and inoculated on BHIA (6.1) medium, and cultured at 30°C ±1°C for 18 h~24 h. If it's flat If there are less than 5 suspicious colonies on the plate, all should be picked. 8.3.2 Extraction of colony protein 8.3.2.1 Take an appropriate amount of bacterial culture (5 mg~10 mg), resuspend the bacterial cells in 300 µL of water, mix well; add 900 µL Absolute ethanol (6.3), vortex and shake to mix; 13 000 g, centrifuge for 2 min, discard the supernatant, centrifuge for 30 s under the same conditions, use a pipette Remove the remaining supernatant and leave it at room temperature for 5 min to allow the ethanol to evaporate. 8.3.2.2 Add 30 µL~50 µL 70% formic acid (6.4) (the amount of formic acid can be adjusted according to the amount of bacterial precipitation), mix well, Add an equal volume of acetonitrile (6.2), vortex to mix; 13,000 g, centrifuge for 2 min, and use the supernatant for spotting. 8.4 Spot Take 1 μL of the supernatant prepared in 8.3.2.2 and spot it on the target plate. At the same time, spot the BTS standard (6.7) on the target plate and place it at room temperature. After the droplets are dried, cover the sample with 1 μL of CHCA matrix solution (6.10), and wait for the CHCA matrix solution (6.10) to dry. MALDI-TOF MS detection. 8.5 MALDI-TOF MS detection 8.5.1 Setting of instrument parameters Select linear operation mode, positive ion mode; detection range. 2 000 Da~20 000 Da; laser hits. 40 times per spectrum (6 Secondary laser accumulation); laser frequency. 60.0 Hz; ion source acceleration voltage. 20 kV. 8.5.2 Instrument calibration Before collecting mass spectrometry data for suspicious colonies, the mass-to-charge ratio of the instrument should be calibrated with BTS standard (6.7) to ensure The error range of the mass-to-charge ratio is less than 0.030 0%. 8.5.3 Data collection and analysis Mass spectrometry data was collected and saved for suspicious colonies, and analyzed and identified by Biotyper software. 8.5.4 Results Judgment Criteria The MALDI-TOF MS identification result gives the 10 strains in the database that most closely match the identified strains, and gives the corresponding Match score. The score is between 2.300 and 3.000, indicating that the credibility of strain identification is very high; between 2.000 and 2.299, indicating that the credibility Identification of genus and possible species; between 1.700 and 1.999, indicating possible identification of genus; between 0.000 and 1.699, indicating Unreliable identification results.

9 Results judgment and report

9.1 The suspicious colony is identified as Bacillus cereus by MALDI-TOF MS, and the matching score is greater than or equal to 1.700, the test result can be determined If it is positive and suspicious, it should be further confirmed and reported according to the method of GB 4789.14. 9.2 For results other than 9.1, the test result can be determined to be negative, and the report does not detect Bacillus cereus. 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.