ISO 15216-1:2017
Microbiology of the food chain — Horizontal method for determination of hepatitis A virus and norovirus using real-time RT-PCR — Part 1: Method for quantification
ISO 15216-1:2017 specifies a method for the quantification of levels of HAV and norovirus genogroup I (GI) and II (GII) RNA, from test samples of foodstuffs (soft fruit, leaf, stem and bulb vegetables, bottled water, BMS) or food surfaces. Following liberation of viruses from the test sample, viral RNA is then extracted by lysis with guanidine thiocyanate and adsorption on silica. Target sequences within the viral RNA are amplified and detected by 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, food 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 Double-stranded DNA (dsDNA) control
4.4.3 EC RNA control
4.5 Test results
5 Reagents
5.1 General
5.2 Reagents used as supplied
5.3 Prepared reagents
6 Equipment and consumables
7 Sampling
8 Procedure
8.1 General laboratory requirements
8.2 Virus extraction
8.2.1 Process control virus material
8.2.2 Negative process control
8.2.3 Food surfaces
8.2.4 Soft fruit, leaf, stem and bulb vegetables
8.2.5 Bottled water
8.2.6 Bivalve molluscan shellfish
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 standard curves
9.3 Calculation of RT-PCR inhibition
9.4 Calculation of extraction efficiency
9.5 Sample quantification
10 Expression of results
11 Precision
11.1 Interlaboratory study
11.2 Repeatability
11.3 Reproducibility limit
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 NucliSENS® system
Annex G (informative) Generation of dsDNA control stocks
Annex H (informative) Generation of EC RNA stocks
Annex I (informative) Typical optical plate layout
Annex J (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 Double-stranded DNA (dsDNA) control
4.4.3 EC RNA control
4.5 Test results
5 Reagents
5.1 General
5.2 Reagents used as supplied
5.3 Prepared reagents
6 Equipment and consumables
7 Sampling
8 Procedure
8.1 General laboratory requirements
8.2 Virus extraction
8.2.1 Process control virus material
8.2.2 Negative process control
8.2.3 Food surfaces
8.2.4 Soft fruit, leaf, stem and bulb vegetables
8.2.5 Bottled water
8.2.6 Bivalve molluscan shellfish
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 standard curves
9.3 Calculation of RT-PCR inhibition
9.4 Calculation of extraction efficiency
9.5 Sample quantification
10 Expression of results
11 Precision
11.1 Interlaboratory study
11.2 Repeatability
11.3 Reproducibility limit
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 NucliSENS® system
Annex G (informative) Generation of dsDNA control stocks
Annex H (informative) Generation of EC RNA stocks
Annex I (informative) Typical optical plate layout
Annex J (informative) Method validation studies and performance characteristics
Bibliography