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)
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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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Sisällysluettelo
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
4.5 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
8.3 RNA extraction
8.4 Real-time RT-PCR
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 Diagram of procedure (normative)
Annex B Composition and preparation of reagents and buffers (normative)
Annex C Real-time RT-PCR mastermixes and cycling parameters (informative)
Annex D Real-time RT-PCR primers and hydrolysis probes for the detection of HAV, norovirus GI and GII and mengo virus (process control) (informative)
Annex E Growth of mengo virus strain MC0 for use as a process control (informative)
Annex F RNA extraction using the NucliSENS® system (informative)
Annex G Generation of dsDNA control stocks (informative)
Annex H Generation of EC RNA stocks (informative)
Annex I Typical optical plate layout (informative)
Annex J Method validation studies and performance characteristics (informative)
ISO 20838:2006 Microbiology of food and animal feeding stuffs -- Polymerase chain reaction (PCR) for the detection of food-borne pathogens -- Requirements for amplification and detection for qualitative methods
ISO 22119:2011 Microbiology of food and animal feeding stuffs -- Real-time polymerase chain reaction (PCR) for the detection of food-borne pathogens -- General requirements and definitions
ISO 22174:2005 Microbiology of food and animal feeding stuffs -- Polymerase chain reaction (PCR) for the detection of food-borne pathogens -- General requirements and definitions
ISO 20838:2006 Microbiology of food and animal feeding stuffs -- Polymerase chain reaction (PCR) for the detection of food-borne pathogens -- Requirements for amplification and detection for qualitative methods
ISO 22119:2011 Microbiology of food and animal feeding stuffs -- Real-time polymerase chain reaction (PCR) for the detection of food-borne pathogens -- General requirements and definitions
ISO 22174:2005 Microbiology of food and animal feeding stuffs -- Polymerase chain reaction (PCR) for the detection of food-borne pathogens -- General requirements and definitions
ISO 7218:2007 Microbiology of food and animal feeding stuffs -- General requirements and guidance for microbiological examinations
882/2004 Rehu- ja elintarvikelainsäädännön sekä eläinten terveyttä ja hyvinvointia koskevien sääntöjen mukaisuuden varmistamiseksi suoritetusta virallisesta valvonnasta