ISO 21872-1:2017
Microbiology of the food chain — Horizontal method for the determination of Vibrio spp. — Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus
ISO 21872-1:2017 specifies a horizontal method for the detection of enteropathogenic Vibrio spp., which causes human illness in or via the intestinal tract. The species detectable by the methods specified include Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus.
ISO 21872-1:2017 is applicable to the following:
- products intended for human consumption and the feeding of animals;
- environmental samples in the area of food production and food handling.
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Foreword
Introduction
1 Scope
2 Normative references
3 Terms and definitions
4 Principle
4.1 General
4.2 Primary enrichment in a liquid selective medium
4.3 Secondary enrichment in a liquid selective medium
4.4 Isolation and identification
4.5 Confirmation
5 Culture media and reagents
5.1 Enrichment medium: alkaline saline peptone water (ASPW)
5.2 Solid selective isolation media
5.2.1 First medium: thiosulphate, citrate, bile and sucrose agar medium (TCBS)
5.2.2 Second medium
5.3 Saline nutrient agar (SNA)
5.4 Reagent for detection of oxidase
5.5 Biochemical tests
5.5.1 L-lysine decarboxylase saline medium (LDC)
5.5.2 Arginine dihydrolase saline medium (ADH)
5.5.3 Reagent for detection of β-galactosidase
5.5.4 Saline medium for detection of indole
5.5.5 Saline peptone waters
5.5.6 Sodium chloride solution
5.6 PCR
5.6.1 Tris acetate EDTA buffer (TAE) (or a buffer allowing similar performance for the purpose)
5.6.2 Mastermix
5.6.3 Primers and probes
5.6.4 Positive control material
5.6.5 Negative extraction control
6 Equipment and consumables
7 Sampling
8 Preparation of the test sample
9 Procedure (See Figure A.1)
9.1 Test portion and initial suspension
9.2 Primary selective enrichment
9.3 Secondary selective enrichment
9.4 Isolation and identification
9.5 Confirmation
9.5.1 General
9.5.2 Selection of colonies for confirmation and preparation of pure cultures
9.5.3 Tests for presumptive identification
9.5.4 Biochemical confirmation
9.5.5 PCR confirmation
9.5.6 DNA extraction
9.5.7 Conventional PCR
9.5.8 Real-time PCR
10 Expression of results
11 Performance characteristics of the method
11.1 Interlaboratory study
11.2 Sensitivity
11.3 Specificity
11.4 LOD50
12 Test report
Annex A (normative) Diagram of procedure
Annex B (normative) Composition and preparation of the culture media and reagents
Annex C (informative) Conventional PCR for the detection of Vibrio parahaemolyticus, thermostable direct haemolysin (tdh) and thermostable direct related haemolysin (trh) genes, Vibrio cholerae and Vibrio vulnificus
Annex D (informative) Real-time PCR for the detection of Vibrio parahaemolyticus, thermostable direct haemolysin gene (tdh) and Vibrio vulnificus
Annex E (informative) Results of an interlaboratory study
Bibliography
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Foreword
Introduction
1 Scope
2 Normative references
3 Terms and definitions
4 Principle
4.1 General
4.2 Primary enrichment in a liquid selective medium
4.3 Secondary enrichment in a liquid selective medium
4.4 Isolation and identification
4.5 Confirmation
5 Culture media and reagents
5.1 Enrichment medium: alkaline saline peptone water (ASPW)
5.2 Solid selective isolation media
5.2.1 First medium: thiosulphate, citrate, bile and sucrose agar medium (TCBS)
5.2.2 Second medium
5.3 Saline nutrient agar (SNA)
5.4 Reagent for detection of oxidase
5.5 Biochemical tests
5.5.1 L-lysine decarboxylase saline medium (LDC)
5.5.2 Arginine dihydrolase saline medium (ADH)
5.5.3 Reagent for detection of β-galactosidase
5.5.4 Saline medium for detection of indole
5.5.5 Saline peptone waters
5.5.6 Sodium chloride solution
5.6 PCR
5.6.1 Tris acetate EDTA buffer (TAE) (or a buffer allowing similar performance for the purpose)
5.6.2 Mastermix
5.6.3 Primers and probes
5.6.4 Positive control material
5.6.5 Negative extraction control
6 Equipment and consumables
7 Sampling
8 Preparation of the test sample
9 Procedure (See Figure A.1)
9.1 Test portion and initial suspension
9.2 Primary selective enrichment
9.3 Secondary selective enrichment
9.4 Isolation and identification
9.5 Confirmation
9.5.1 General
9.5.2 Selection of colonies for confirmation and preparation of pure cultures
9.5.3 Tests for presumptive identification
9.5.4 Biochemical confirmation
9.5.5 PCR confirmation
9.5.6 DNA extraction
9.5.7 Conventional PCR
9.5.8 Real-time PCR
10 Expression of results
11 Performance characteristics of the method
11.1 Interlaboratory study
11.2 Sensitivity
11.3 Specificity
11.4 LOD50
12 Test report
Annex A (normative) Diagram of procedure
Annex B (normative) Composition and preparation of the culture media and reagents
Annex C (informative) Conventional PCR for the detection of Vibrio parahaemolyticus, thermostable direct haemolysin (tdh) and thermostable direct related haemolysin (trh) genes, Vibrio cholerae and Vibrio vulnificus
Annex D (informative) Real-time PCR for the detection of Vibrio parahaemolyticus, thermostable direct haemolysin gene (tdh) and Vibrio vulnificus
Annex E (informative) Results of an interlaboratory study
Bibliography