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SN/T 5228.8-2019: (Rapid screening method for pathogenic microorganisms in exported foods MALDI-TOF MS method Part 8: Klebsiella pneumoniae)
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(Rapid screening method for pathogenic microorganisms in exported foods MALDI-TOF MS method Part 8: Klebsiella pneumoniae)
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Basic data | Standard ID | SN/T 5228.8-2019 (SN/T5228.8-2019) | | Description (Translated English) | (Rapid screening method for pathogenic microorganisms in exported foods MALDI-TOF MS method Part 8: Klebsiella pneumoniae) | | Sector / Industry | Commodity Inspection Standard (Recommended) | | Classification of Chinese Standard | C53 | | Classification of International Standard | 67.050 | | Word Count Estimation | 8,886 | | Date of Issue | 2019 | | Date of Implementation | 2020-07-01 | | Issuing agency(ies) | General Administration of Customs | SN/T 5228.8-2019: (Rapid screening method for pathogenic microorganisms in exported foods MALDI-TOF MS method Part 8: Klebsiella pneumoniae)
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Rapid detection of pathogens in export food-MALDI-TOF MS method-
Part 8.Klebsiella pneumoniae
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 8 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 organizations of this section. Hangzhou Customs of the People's Republic of China, Beijing Customs of the People's Republic of China.
The main drafters of this section. Li Ke, Fang Ying, Shen Biao, Lu Jinhu, Zeng Jing, Wang Qi.
MALDI-TOF MS method for rapid screening of pathogenic microorganisms in exported food
1 Scope
This part of SN/T 5228 specifies the MALDI-TOF MS rapid screening method for Klebsiella pneumoniae in exported food.
This section applies to the rapid screening of Klebsiella pneumoniae in exported 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/T 14926.13 Test method for Klebsiella pneumoniae in laboratory animals
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, Klebsiella pneumoniae should be tested by qualified personnel, and all cultures and waste should be tested for Klebsiella pneumoniae.
Discarded materials should 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 Buffered peptone water (BPW). see Appendix A, Chapter A.1.
6.3 Cholestyrol Agar (DHL). See Appendix A, Chapter A.2.
6.4 Acetonitrile (ACN).
6.5 Anhydrous ethanol.
6.6 Formic acid.
6.7 Trifluoroacetic acid (TFA).
6.8 CHCA.
6.9 BTS standard product.
6.10 Solvent I. Prepared according to the ratio of water. acetonitrile (6.4). trifluoroacetic acid (6.7) to 50.47.5.2.5 (volume ratio).
Now use.
6.11 Preparation of BTS standard solution. Dissolve BTS standard (6.9) with 50 µL solvent I (6.10). Pipette repeatedly to dissolve
BTS powder (avoid vigorous shaking), leave it at room temperature for 5 minutes, and dissolve again. After all BTS (6.9) is dissolved, centrifuge it immediately and use
200 µL PCR tubes are aliquoted, 5 µL per tube, stored at -20°C for later use.
6.12 Preparation of CHCA matrix solution. Add solvent I (6.10) to CHCA (6.8) 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
8.1 Detection flow chart
The MALDI-TOF MS detection process of Klebsiella pneumoniae in food is shown in Figure 1.
8.2 Sample preparation, enrichment culture
Weigh 25 g of the sample into a sterile homogenization cup containing 225 mL BPW, and homogenize for 1 min to 2 min at 8 000 r/min~10 000 r/min.
Or put it in a sterile homogenization bag containing 225 mL BPW, and beat it with a flapping homogenizer for 1 min~2 min. If the sample is liquid, draw 25
Put the mL sample into a sterile Erlenmeyer flask containing 225 mL BPW (an appropriate number of sterile glass beads can be pre-installed in the flask), shake and mix well. in
Incubate at 36°C ±1°C for 18 h~24 h.
8.3 Separation
Streak the enriched culture on DHL (6.3) plates and incubate at 36℃±1℃ for 18 h~24 h. Observe each level.
Morphology of colonies growing on the plate. The Klebsiella pneumoniae colonies on the DHL (6.3) plate are pale pink, large and raised, smooth and moist.
Mucus-like, adjacent colonies easily fuse into pus-like, filamentous adhesions when the colonies are picked by the inoculation needle.
8.4 Treatment of suspicious colonies
Either the direct smear method or the formic acid extraction method can be used for suspicious colony treatment. If you can't get a high-quality picture by directly painting
Spectrum, the formic acid extraction method should continue to be used to treat suspicious colonies.
8.4.1 Direct application method
Use a toothpick or a 10 µL pipette tip to directly pick up the suspicious colony from the selective plate and smear it on the target plate to form a uniform thin layer (the thinner the more
it is good). Cover with 1 μL of CHCA matrix solution (6.12), and perform MALDI-TOF MS detection after the CHCA matrix solution (6.12) is dried.
8.4.2 Formic acid extraction method
8.4.2.1 Inoculation culture
At least 5 suspicious colonies should be picked, streaked and inoculated on a blood agar plate (6.1), and cultured at 36°C ± 1°C for 18 h~24 h. If on the tablet
If there are less than 5 suspicious colonies, all should be picked.
8.4.2.2 Extraction of colony protein
8.4.2.2.1 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 (6.5), vortex and shake; 13 000 g, centrifuge for 2 min, discard the supernatant, centrifuge for 30 s under the same conditions, and pipette
Remove the remaining supernatant, and place it at room temperature for 5 min to allow the ethanol to evaporate.
8.4.2.2.2 Add 30 µL~50 µL 70% formic acid (6.6) (the amount of formic acid can be adjusted according to the amount of bacterial precipitation), mix well,
Add an equal volume of acetonitrile (6.4), vortex and mix; centrifuge at 13,000 g for 2 min, and use the supernatant for spotting.
8.5 Spot
Take 1 μL of the supernatant prepared in 8.4.2.2.2 and spot it on the target plate. At the same time, spot the BTS standard (6.9) 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 (6.12), and wait for the CHCA matrix solution (6.12) to dry.
MALDI-TOF MS detection.
8.6 MALDI-TOF MS detection
8.6.1 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.
8.6.2 Instrument calibration
Before collecting mass spectrometry data for suspicious colonies, the mass-to-charge ratio of the instrument should be calibrated with the BTS standard (6.9) to ensure
The error range of the mass-to-charge ratio is less than 0.030 0%.
8.6.3 Data collection and analysis
Mass spectrometry data was collected and saved for suspicious colonies, and analyzed and identified by Biotyper software.
8.6.4 Results Judgment Standard
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 Klebsiella pneumoniae by MALDI-TOF MS, and the matching score is greater than or equal to 1.700, the test result can be determined
If the result is suspicious, it should be confirmed and reported according to the method of GB/T 14926.13.
9.2 For results other than 9.1, the test result can be determined to be negative, and no Klebsiella pneumoniae is reported.
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.
Appendix A
(Normative appendix)
Media and reagents
A.1 Buffered peptone water (BPW)
A.1.2 Preparation method
Heat and dissolve the ingredients in A.1.1, adjust the pH to 7.2±0.2 at 25°C, and dispense into 500 mL jars, each with 225 mL,
Autoclave at 121°C for 15 minutes.
A.2 Cholestyrol Agar (DHL)
A.2.2 Preparation method
In addition to neutral red and agar, add other ingredients to 400 mL of distilled water, stir evenly, let stand for about 10 minutes, heat and boil until
completely dissolved. Adjust the pH to 7.3±0.1, then add the agar to 600 mL of distilled water, stir evenly, let stand for about 10 minutes, add
Bring to a boil until completely dissolved. After mixing the two solutions, add 6 mL of 5 g/L neutral red aqueous solution, stir evenly, and wait to cool to
50°C ~55°C, pour into a plate for use
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