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BSI PD CEN/TS 17711:2022

BSI PD CEN/TS 17711:2022

$167.15

Plant biostimulants. Detection of Vibrio spp.

Published By Publication Date Number of Pages
BSI 2022 36
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This document specifies a horizontal method for the detection of enteropathogenic Vibrio spp., which causes human illness in or via the intestinal tract [1]. The species detectable by the methods specified include Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. It is applicable to the following: — microbial plant biostimulants. NOTE 1 The World Health Organization (WHO) has identified that V. parahaemolyticus, V. cholerae and V. vulnificus are the major contaminants of Vibrio spp. [1]. NOTE 2 For confirmation, it is possible to use PCR tests; in this case the laboratory validates the procedure and data generated.

PDF Catalog

PDF Pages PDF Title
2 undefined
7 1 Scope
2 Normative references
3 Terms and definitions
8 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
9 4.5 Confirmation
5 Culture media and reagents
10 6 Equipment and consumables
11 7 Sampling
8 Preparation of the test sample
9 Procedure (see Figure A.1)
9.1 Test portion and initial suspension
12 9.2 Primary selective enrichment
9.3 Secondary selective enrichment
13 9.4 Isolation and identification
9.5 Confirmation
9.5.1 General
14 9.5.2 Selection of colonies for confirmation and preparation of pure cultures
9.5.3 Tests for presumptive identification
9.5.3.1 Oxidase test
9.5.3.2 Microscopic examination (optional)
9.5.3.3 Selection of the cultures
9.5.4 Biochemical confirmation
9.5.4.1 General
15 9.5.4.2 L-lysine decarboxylase saline medium (5.5.1)
9.5.4.3 Arginine dihydrolase saline medium (5.5.2)
9.5.4.4 Detection of β-galactosidase (5.5.3)
9.5.4.5 Detection of indole (5.5.4)
9.5.4.6 Halotolerance test (5.5.5)
9.5.4.7 Interpretation of biochemical tests
16 9.5.4.8 Step by step confirmation (optional)
10 Expression of results
11 Performance characteristics of the method
11.1 Sensitivity
17 11.2 Specificity
11.3 LOD50
12 Test report
18 Annex A (normative)Diagram of procedure
19 Annex B (normative)Composition and preparation of the culture media and reagents
B.1 Introduction
B.2 Water
B.3 Alkaline saline peptone water (ASPW)
B.3.1 Composition
B.3.2 Preparation
20 B.4 Thiosulfate citrate bile and sucrose agar (TCBS)
B.4.1 Composition
B.4.2 Preparation
B.4.3 Preparation of the agar dishes
B.5 Saline nutrient agar (SNA)
B.5.1 Composition
B.5.2 Preparation
21 B.5.3 Preparation of the agar dishes
B.5.4 Preparation of slants of saline nutrient agar
B.6 Reagent for detection of oxidase
B.6.1 Composition
B.6.2 Preparation
B.7 L-lysine decarboxylase saline medium (LDC)
B.7.1 Composition
B.7.2 Preparation
B.8 Arginine dihydrolase saline medium (ADH)
B.8.1 Composition
22 B.8.2 Preparation
B.9 Detection of β-galactosidase
B.9.1 ONPG solution
B.9.1.1 Composition
B.9.1.2 Preparation
B.9.2 Buffer solution
B.9.2.1 Composition
B.9.2.2 Preparation
B.9.3 Complete reagent
B.9.3.1 Composition
B.9.3.2 Preparation
23 B.10 Saline medium for detection of indole
B.10.1 Tryptophan saline medium
B.10.1.1 Composition
B.10.1.2 Preparation
B.10.2 Kovacs reagent
B.10.2.1 Composition
B.10.2.2 Preparation
B.11 Saline peptone water
B.11.1 Composition
B.11.2 Preparation
24 B.12 Sodium chloride solution
B.12.1 Composition
B.12.2 Preparation
B.13 Tris acetate EDTA (TAE) buffer
B.13.1 Composition
B.13.2 Preparation
25 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
C.1 General
C.2 Equipment
C.2.1 Tris acetate EDTA buffer (TAE) (or a buffer allowing similar performance for the purpose)
C.2.2 Mastermix
C.2.3 Composition
26 C.3 DNA extraction
C.4 Procedure Conventional PCR
27 C.5 Primers and probes
C.5.1 General
C.5.2 Vibrio parahaemolyticus primers (conventional PCR)
28 C.5.3 Thermostable direct haemolysin (tdh) and thermostable direct related haemolysin (trh) genes Vibrio parahaemolyticus primers (conventional PCR)
C.5.4 Vibrio cholerae primers (conventional PCR)
C.5.5 Vibrio vulnificus primers (conventional PCR)
C.5.6 Cycling parameters — VptoxR and VVH
29 C.5.7 Cycling parameters — prVC
C.5.8 Cycling parameters — tdh and trh
C.6 Control material — conventional PCR
31 Annex D (informative)Real-time PCR for the detection of Vibrio parahaemolyticus, thermostable direct haemolysin gene (tdh) and Vibrio vulnificus
D.1 General
D.2 Equipment
D.2.1 Tris acetate EDTA buffer (TAE) (or a buffer allowing similar performance for the purpose)
D.2.2 Mastermix
D.2.3 Composition of mastermix
D.3 DNA extraction
D.4 Real-time PCR
32 D.5 Vibrio parahaemolyticus — primers and hydrolysis probes
D.6 Thermostable direct haemolysin (tdh) gene Vibrio parahaemolyticus — primers and hydrolysis probes
33 D.7 Vibrio vulnificus — primers and hydrolysis probes
D.8 Cycling parameters
D.9 Control material – real-time PCR

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BSI PD CEN/TS 17711:2022
$167.15