ISO 15216-2:2019
Microbiology of the food chain — Horizontal method for determination of hepatitis A virus and norovirus using real-time RT-PCR — Part 2: Method for detection
This document specifies a method for detection of hepatitis A virus (HAV) and norovirus genogroups I (GI) and II (GII), from test samples of foodstuffs [(soft fruit, leaf, stem and bulb vegetables, bottled water, bivalve molluscan shellfish (BMS)] or surfaces using real-time RT-PCR.
This method is not validated for detection of the target viruses in other foodstuffs (including multi-component foodstuffs), or any other matrices, nor for the detection of other viruses in foodstuffs, surfaces or other matrices.
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Foreword
Introduction
1 Scope
2 Normative references
3 Terms and definitions
4 Principle
4.1 Virus extraction
4.2 RNA extraction
4.3 Real-time RT-PCR
4.4 Control materials
4.4.1 Process control virus
4.4.2 EC RNA control
4.5 Test results
5 Reagents
5.1 General
5.2 Reagents used as supplied
5.3 Reagents requiring preparation
6 Equipment and consumables
7 Sampling
8 Procedure
8.1 General laboratory requirements
8.2 Virus extraction
8.2.1 General
8.2.2 Process control virus material
8.2.3 Negative process control
8.2.4 Surfaces
8.2.5 Soft fruit and leaf, stem and bulb vegetables
8.2.6 Bottled water
8.2.7 Bivalve molluscan shellfish (BMS)
8.3 RNA extraction
8.4 Real-time RT-PCR
8.4.1 General requirements
8.4.2 Real-time RT-PCR analysis
9 Interpretation of results
9.1 General
9.2 Construction of process control virus RNA standard curve
9.3 Control for RT-PCR inhibition
9.4 Calculation of extraction efficiency
10 Expression of results
11 Performance characteristics of the method
11.1 Validation 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 reagents and buffers
Annex C (informative) Real-time RT-PCR mastermixes and cycling parameters
Annex D (informative) Real-time RT-PCR primers and hydrolysis probes for the detection of HAV, norovirus GI and GII and mengo virus (process control)
Annex E (informative) Growth of mengo virus strain MC0 for use as a process control
Annex F (informative) RNA extraction using the BioMerieux NucliSens® system
Annex G (informative) Generation of external control RNA (EC RNA) stocks
Annex H (informative) Typical optical plate layout
Annex I (informative) Method validation studies and performance characteristics
Bibliography
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Foreword
Introduction
1 Scope
2 Normative references
3 Terms and definitions
4 Principle
4.1 Virus extraction
4.2 RNA extraction
4.3 Real-time RT-PCR
4.4 Control materials
4.4.1 Process control virus
4.4.2 EC RNA control
4.5 Test results
5 Reagents
5.1 General
5.2 Reagents used as supplied
5.3 Reagents requiring preparation
6 Equipment and consumables
7 Sampling
8 Procedure
8.1 General laboratory requirements
8.2 Virus extraction
8.2.1 General
8.2.2 Process control virus material
8.2.3 Negative process control
8.2.4 Surfaces
8.2.5 Soft fruit and leaf, stem and bulb vegetables
8.2.6 Bottled water
8.2.7 Bivalve molluscan shellfish (BMS)
8.3 RNA extraction
8.4 Real-time RT-PCR
8.4.1 General requirements
8.4.2 Real-time RT-PCR analysis
9 Interpretation of results
9.1 General
9.2 Construction of process control virus RNA standard curve
9.3 Control for RT-PCR inhibition
9.4 Calculation of extraction efficiency
10 Expression of results
11 Performance characteristics of the method
11.1 Validation 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 reagents and buffers
Annex C (informative) Real-time RT-PCR mastermixes and cycling parameters
Annex D (informative) Real-time RT-PCR primers and hydrolysis probes for the detection of HAV, norovirus GI and GII and mengo virus (process control)
Annex E (informative) Growth of mengo virus strain MC0 for use as a process control
Annex F (informative) RNA extraction using the BioMerieux NucliSens® system
Annex G (informative) Generation of external control RNA (EC RNA) stocks
Annex H (informative) Typical optical plate layout
Annex I (informative) Method validation studies and performance characteristics
Bibliography