🦠 Screening detection of staphylococcal enterotoxins SEA, SEB, SEC, SED and SEE in foodstuffs — 25 g of sample with about 40 ml of water at 38 °C is homogenized, shaken for 30–60 min, acidified to pH 3.5–4.0, centrifuged (≥ 3130 g, 15 min), neutralized to pH 7.4–7.6, the extract is concentrated by dialysis in PEG solution to 5.0–5.5 g, and the toxins are detected with a commercial immunoenzymatic kit (e.g. ELFA, ELISA) meeting the criteria: sensitivity and specificity above 90 %, LOD50 below 0.06 ng/g; concentration mandatory for milk and dairy products, PN-EN ISO 19020

Microbiology PN-EN ISO 19020

Overview

WHAT THE STANDARD COVERS. We read the text of the standard in the iTeh sample of ISO 19020:2017 (English text): title page, contents, foreword, introduction, Clauses 1–8 and the beginning of Clause 9 (quality control) — the sample ends on page 6. Subclause 8.7 “Detection” is truncated in the sample (two sentences remain on choosing the kit and following the manufacturer’s instructions), and in Clause 2 part of the first entry is missing (ISO 3696, water for analysis, known from 5.1). We do not read Clauses 10–13 (expression of results, confirmation, performance characteristics, test report) or Annexes A–C; we know their titles from the contents.

ACCORDING TO THE TEXT OF ISO 19020:2017. The document was prepared by CEN/TC 275 “Food analysis — Horizontal methods” in collaboration with ISO/TC 34 “Food products”, Subcommittee SC 9 “Microbiology” (Vienna Agreement). Contents: 8 Procedure (8.1 test portion, 8.2 storage of the test sample, 8.3 extraction, 8.4 concentration of the extract — mandatory for milk and dairy products, 8.5 recovery of the concentrated extract, 8.6 storage before detection, 8.7 detection, 8.8 performance criteria), 9 Quality control, 10 Expression of results, 11 Confirmation, 12 Performance characteristics of the method, 13 Test report, Annexes A and B (informative) — results of interlaboratory studies of 2013 and 2014, Annex C (informative) “Note on interferences”. Introduction: staphylococcal enterotoxins (SEs) are proteins produced in food by certain strains of coagulase-positive staphylococci, mainly Staphylococcus aureus; they are heat and acid stable and cause nausea, vomiting, abdominal pain and diarrhoea; because of their stability they may be present even when staphylococci cannot be detected; they form a family of more than 20 proteins of 19–30 kDa, resistant to heat treatment, freezing, pH changes and proteolysis; nanogram amounts can cause intoxication; the European Union has set microbiological criteria including enumeration of coagulase-positive staphylococci and detection of SEs. LC-MS/MS methods are under development, so enzyme immunoassay (EIA) methods with commercial kits were chosen as the starting point of standardization; kit performance criteria were evaluated on five food matrices; the acceptable LOD50 was derived from a benchmark dose (BMD) established by the US EPA methodology from outbreak data, including data of the EU reference laboratory for coagulase-positive staphylococci. Scope (1): screening method for the detection of enterotoxins SEA, SEB, SEC, SED and SEE in foodstuffs, in two steps: extraction with concentration based on dialysis and immunoenzymatic detection with a commercial kit; it applies to products intended for human consumption; other enterotoxins (SEG, SEH, SEI, SER, SES, SET) also cause illness, but for lack of kits the standard covers only SEA–SEE and may apply to others after validation. References (2): ISO 3696 (water for analysis), ISO 7218 (general requirements for microbiological examinations). Definitions (3): SEA–SEE — exoproteins of enterotoxigenic strains with a molecular weight of 19–30 kDa; specificity (SP) — number of negative results divided by the number of blank samples tested; sensitivity (SE) — number of positive results divided by the number of samples tested at a given contamination level; LOD50 — concentration (ng SE/g) at which the probability of detection is 50 %; BMD — dose of a hazard likely to trigger symptoms in a given percentage of the exposed population. Reagents (5): water according to ISO 3696; hydrochloric acid and sodium hydroxide (e.g. 5N, 1N); PBS pH 7.3 ± 0.2 (NaCl/Na2HPO4 145/10 mM); PEG solution of molecular weight 20 000 g/mol (30 g of powder with 70 ml of water); electrode cleaning solution (e.g. ethanol 70 %); immunoenzymatic SE detection kit meeting the criteria of 8.8. Apparatus (6): microbiological equipment according to ISO 7218; blender; balance; homogenizer — a rotary homogenizer is highly recommended, with a peristaltic homogenizer only bags without filter; shaker; pH meter; centrifuge ≥ 3130 g, preferably refrigerated; dialysis membrane with a molecular weight cut-off of 6000–8000 Da with closures; filtering material; shallow tray; refrigerator (3 ± 2) °C or (5 ± 3) °C and freezer ≤ −18 °C; glass or polypropylene ware to avoid adsorption of toxins; water bath (38 ± 2) °C. Sampling (7): not part of the method. Test portion (8.1): for cheese with rind about 10 % rind and 90 % core; toxins may be heterogeneously distributed, so the whole sample or a representative part is homogenized and 25 g taken; in a suspected food poisoning outbreak the portion may be smaller, and the ratio of test portion mass to concentrated extract mass should be about 5 (25 g for 5.0–5.5 g extract, max. 5.8 g for sticky extracts). Storage (8.2): recommended at (3 ± 2) °C or (5 ± 3) °C; without analysis within 24 h — freeze, thaw completely in the refrigerator, avoid repeated freezing. Extraction (8.3): about 40 ml of water at 38 °C added to 25 g (liquid products without water), homogenization (particularly important for fatty products); shaking 30–60 min at 18–27 °C; acidification with HCl to pH 3.5–4.0; centrifugation ≥ 3130 g for 15 min at about 4 °C or at room temperature (refrigeration recommended for fatty samples); after centrifugation the pH must be 3.0–4.5 — above 4.5 acidify again, below 3.0 the structure of SEs may be damaged and a new portion must be taken; neutralization with NaOH to pH 7.4–7.6 — above 9.0 a new portion; centrifugation again and recovery of the entire aqueous phase. Variant without dialysis (8.3.10): only in limited circumstances, e.g. a suspected food poisoning event, and not for milk and milk products — with a negative result the rest of the phase is concentrated the same day and detection repeated. Concentration (8.4): membrane rehydrated and rinsed, filled with the entire neutralized phase through a funnel with filter material, laid in a shallow tray with PEG solution overnight in the refrigerator; if more than 5 ml remains in the membrane — longer (up to 3 days) or with added PEG powder. Recovery (8.5): the membrane is rinsed free of PEG, the extract is recovered with PBS (milk and milk products) or water (other matrices), rubbing the inner surfaces, to a mass of 5.0–5.5 g (5.8 g) per 25 g portion. Storage of the extract (8.6): up to 48 h in the refrigerator, longer at ≤ −18 °C. Detection (8.7, truncated): a kit meeting the criteria for the entire procedure, following the manufacturer’s instructions. Criteria (8.8): specificity and sensitivity above 90 %, LOD50 below 0.06 ng SE/g — from the BMD for SEA of 6.1 ng and ingestion of 100 g of food; for lack of data for other types this value was also adopted for SEB–SEE; values for different kits and matrices with and without dialysis are given in Clause 12, and laboratories are to select the kit on that basis; results of interlaboratory studies may not apply to food types other than those in Annexes A and B. Quality control (9, beginning): checking the entire procedure with reference materials is recommended.

STATUS (as of 03.10.2026). In the PKN search (query “19020”, 03.10.2026) the only result is PN-EN ISO 19020:2017-08 (English version, “Wprowadza: EN ISO 19020:2017 [IDT], ISO 19020:2017 [IDT]”), with no withdrawal note. Card: publication date 23.08.2017, 36 pages, KT 3 Mikrobiologii Łańcucha Żywnościowego, ICS 07.100.30, no withdrawal note. In the iTeh catalogue (read 03.10.2026) ISO 19020:2017 has the status “Published”, stage 90.93 (International Standard confirmed).

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, data up to 17.09.2026, read 03.10.2026). The number 19020 appears in 4 records at 4 laboratories: AB 377, AB 485, AB 486, AB 598. In the current PCA documents (read 03.10.2026; AB 377 issue no. 29 of 12.12.2025, AB 486 issue no. 29 of 23.07.2026, AB 598 issue no. 36 of 30.07.2026, AB 485 issue no. 27 of 19.08.2026) the number appears in 4 lines at the same 4 laboratories; in each document the number of page markers equals the number of PDF pages, without repetitions; a search of all PCA documents downloaded from the BIP on 03.10.2026 found no laboratories outside the database copy. Form “PN-EN ISO 19020:2017-08” (AB 485), “PN-EN ISO 19020: 2017-08 z wył. pkt 11” (AB 377), “PN-EN ISO 19020 z wyłączeniem pkt. 11” with the instruction of the Vidas Staph Enterotoxin II (SET2) kit (AB 598) and “PN-EN ISO 19020” (AB 486). None of these items is suspended. The “Laboratories” tab (query “PN-EN ISO 19020”) shows all 4 laboratories — checked by a tab query on the labcoda.pl production server on 03.10.2026.

WHAT THE LABORATORIES TEST (items in PCA). Object: raw milk and raw-milk products, heat-treated milk and dairy products, raw meat and meat products and poultry ready to cook, ready-to-eat and ready-to-reheat meat products and other categories (AB 377); meat and meat products, milk and dairy products, fish and fish products, eggs and egg products, ready meals (AB 485); food (AB 486, AB 598). Characteristic: presence of staphylococcal enterotoxins. Technique: enzyme-linked fluorescent assay (ELFA) with a mini VIDAS instrument (AB 377), ELFA (AB 598), immunoenzymatic with a mini VIDAS instrument (AB 486), ELFA or ELISA (AB 485).

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. Negative result: the standard detects only SEA–SEE, while other enterotoxins (SEG, SEH, SEI, SER, SES, SET) also cause illness (1) — a negative result does not exclude toxins outside these five. Kit: the result depends on the kit, which for the entire procedure, including extraction, must meet LOD50 < 0.06 ng/g and sensitivity and specificity > 90 % (8.8); the data in Clause 12 concern the matrices of the interlaboratory studies. pH: below 3.0 after acidification or above 9.0 after neutralization the toxin structure may be damaged and the portion must be repeated (8.3.6, 8.3.7). Milk: concentration by dialysis is mandatory and the variant without dialysis is not allowed (8.3.10, 8.4). Ware: glass or polypropylene, because toxins adsorb to other materials (6.12). Sample: repeated freezing and thawing causes toxin losses (8.2). Exclusion: AB 377 and AB 598 exclude clause 11, which according to the contents concerns confirmation — we have not read the content of that clause.

WHAT WE DO NOT GIVE. Expression of results, the confirmation procedure, performance data for individual kits and matrices, interlaboratory results or notes on interferences (Clauses 10–13, Annexes A–C) are not quoted — they lie outside the sample. We do not give microbiological criteria from regulations.

Method principle

From 25 g of sample (with about 40 ml of water at 38 °C) the toxins are extracted by shaking, acidified to pH 3.5–4.0, centrifuged, neutralized to pH 7.4–7.6 and centrifuged again. The aqueous phase is concentrated by dialysis in a 6000–8000 Da membrane immersed in PEG 20 000 solution, and the extract is recovered to 5.0–5.5 g. Enterotoxins SEA–SEE are detected with a commercial immunoenzymatic kit which for the entire procedure meets sensitivity and specificity > 90 % and LOD50 < 0.06 ng/g.

Applications

• Screening detection of staphylococcal enterotoxins SEA, SEB, SEC, SED and SEE in products intended for human consumption (1)
• Suspected food poisoning — smaller portion and variant without dialysis, but not for milk and milk products (8.1, 8.3.10)
• Milk and dairy products, meat, poultry, fish, eggs, ready meals, food in general — items in the accreditation scopes of 4 laboratories in PCA

Key parameters

Standard

Standard number

PN-EN ISO 19020:2017-08

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda immunoenzymatycznego wykrywania enterotoksyn gronkowcowych w żywności

Title (EN)

Microbiology of the food chain — Horizontal method for the immunoenzymatic detection of staphylococcal enterotoxins in foodstuffs

Step-by-step procedure

1. Test portion

   25 g of homogenized sample (cheese: 10 % rind, 90 % core); storage in the refrigerator, no repeated freezing (8.1, 8.2). PN-EN ISO 19020:2017-08 with no withdrawal note in PKN (03.10.2026).

2. Extraction

   About 40 ml water 38 °C, homogenization, shaking 30–60 min (8.3.1–8.3.3).

3. pH clean-up

   Acidification to pH 3.5–4.0, centrifugation, pH check 3.0–4.5, neutralization to 7.4–7.6, centrifugation (8.3.4–8.3.9).

4. Concentration

   Dialysis in PEG overnight in the refrigerator; extract recovered with PBS or water to 5.0–5.5 g (8.4, 8.5).

5. Detection

   Immunoenzymatic kit meeting the criteria of 8.8, according to the manufacturer’s instructions (8.7, 8.8).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Staphylococcal enterotoxins — work in a microbiological laboratory according to ISO 7218, avoid aerosols
• Hydrochloric acid and sodium hydroxide at 5N — corrosive, eye and hand protection

Frequently asked questions