Date: 2027-06

prEN 15634-8

Secretariat: CEN/TC 275

Foodstuffs - Detection of food allergens by molecular biological methods - Part 8: Peanut (Arachis hypogaea), hazelnut (Corylus spp.), walnut (Juglans regia) and cashew (Anacardium occidentale) - Qualitative detection of specific DNA sequences in food by real-time PCR

Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen Verfahren - Teil 8: Erdnuss (Arachis hypogaea), Haselnuss (Corylus spp.), Walnuss (Juglans regia) und Cashew (Anacardium occidentale) - Qualitativer Nachweis spezifischer DNA-Sequenzen in Lebensmitteln mittels Real-time PCR

CCMC will prepare and attach the official title page

European foreword 3

Introduction 4

1 Scope 5

2 Normative references 5

3 Terms and definitions 5

4 Principle 5

5 Reagents 6

5.1 General 6

5.2 Extraction reagents 6

5.3 Real-time PCR reagents 7

5.3.1 Master mix for real-time PCR, containing thermostable DNA polymerase (for hot-start PCR) and PCR buffer solution (containing reaction buffer, dNTPs, MgCl₂), as a dilutable concentrate. 7

5.3.2 Oligonucleotides 7

6 Apparatus and equipment 8

6.1 General 8

6.2 DNA extraction 8

6.3 PCR 9

7 Procedure 9

7.1 General 9

7.2 Sample preparation 9

7.3 Preparation of DNA extracts 9

7.3.1 DNA extraction with CTAB and DNA purification 9

7.3.2 Optional quantification of DNA concentration 10

7.4 Real-time PCR set-up 11

7.4.1 Reaction mix for real-time PCR 11

7.4.2 Positive control for DNA targets 12

7.4.3 Negative control for DNA targets 12

7.4.4 Amplification reagent control 12

7.4.5 Extraction blank control 12

7.4.6 Positive extraction control 12

7.4.7 Temperature/time program (real-time PCR) 12

7.4.8 Accept/Reject criteria 13

7.4.9 Identification 13

8 Validation 13

8.1 General 13

8.2 Specificity 13

8.3 Sensitivity 14

8.4 Method validating interlaboratory study (ring trial) 14

8.4.1 Setup of the ring trial 14

8.4.2 Deviations from the ring trial protocol 16

8.4.3 Ring trial validation results 16

9 Test report 24

Bibliography 25

European foreword

This document (prEN 15634-8:2026) has been prepared by Technical Committee CEN/TC 275 "Food analysis - Horizontal method", the secretariat of which is held by DIN.

This document is currently submitted to the CEN Enquiry.

Introduction

Introduction

For the use of this document the term:

— ‘shall’ indicates a requirement;

— ‘should’ indicates a recommendation;

— ‘may’ indicates a permission; and

— ‘can’ indicates a possibility and/or a capability.

1 Scope

This document specifies a method for the qualitative detection of the species-specific DNA of peanut (Arachis hypogaea), hazelnut (Corylus spp.), walnut (Juglans regia) and cashew (Anacardium occidentale) in food of animal and plant origin, using real-time PCR, in the context of allergen analyses.

The method was previously validated in an interlaboratory study (ring trial) and applied to DNA extracted from samples that consist of defined proportions of peanut, hazelnut, walnut and cashew in rice biscuits, cooked sausage, sauce powder, vegan cookie and veggie burger (powder).

The limit of detection of each real-time PCR has been determined experimentally to be about 5 mg/kg (10 mg/kg for roasted peanuts).

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content constitutes requirements of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.

EN 15634‑1, Foodstuffs - Detection of food allergens by molecular biological methods - Part 1: General considerations

EN 15842, Foodstuffs - Detection of food allergens - General considerations and validation of methods

3 Terms and definitions

For the purposes of this document, the terms and definitions given in EN 15634‑1and EN 15842and the following apply.

ISO and IEC maintain terminology databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at http://www.iso.org/obp

— IEC Electropedia: available at http://www.electropedia.org/

4 Principle

Total DNA is extracted from the sample using a cetyltrimethylammonium bromide (CTAB) method EN ISO 21571 [1]. In general, CTAB functions as a cationic surfactant, leading to the formation of complexes with DNA which are precipitated in the presence of low salt concentration leaving other impurities in solution.

In short, potential PCR inhibitors are removed from the DNA extract through chloroform extraction and subsequent CTAB precipitation followed by an alcoholic precipitation of the DNA. The DNA precipitate is washed, dissolved in buffer solution and the DNA content can be measured.

From the extracted sample DNA, nut-specific DNA sequences are amplified using real-time PCR.

For the different genera the following specific DNA target sequences are used:

— Peanut: multicopy sequence from mitochondrial DNA close to the coding region of the ATPase subunit 6 (atp6) of Arachis hypogaea, 104 base pairs (bp) long (Genbank^1[1]​: MW448460.1 [2][3]

— Hazelnut: multicopy sequence from the internal transcribed spacer 2 (ITS2) region from the 5.8S ribosomal RNA gene of Corylus spp., 62 bp long (GenBank: MG237811.1 [4])

— Walnut: multicopy sequence from a non-coding segment of the large single copy (LSC) region of the chloroplast genome of Juglans regia, 67 bp long (GenBank¹: MF167463.1 [4])

— Cashew: multicopy sequence from the internal transcribed spacer 2 (ITS2) region from the 5.8S ribosomal RNA gene of Anacardium occidentale, 69 bp long (GenBank¹: AB071690.1 [4]).

The real-time PCR method involves a fluorescence detection with sequence-specific hydrolysis probes, see [2].

5 Reagents

5.1 General

The following general conditions for analysis should be followed, unless specified otherwise. Use only analytical-grade reagents suitable for molecular biology. All water shall be free from DNA and nucleases, e.g. double distilled or equivalent (molecular grade). Solutions shall be prepared by dissolving the appropriate reagents in water and autoclaving, unless otherwise specified.

5.2 Extraction reagents

5.2.1  Chloroform.

5.2.2  Ethanol, volume fraction φ = 70 %.

5.2.3  Ethylenediaminetetraacetic acid disodium salt (Na₂EDTA).

5.2.4  Cetyltrimethylammonium bromide (CTAB).

5.2.5  Hydrochloric acid, mass fraction w = 37 %.

5.2.6  Isopropyl alcohol.

5.2.7  Proteinase K.

5.2.8  RNase A.

5.2.9  α-Amylase (>1 500 U/mg protein).

5.2.10  Sodium chloride.

5.2.11  Sodium hydroxide solution.

5.2.12  Tris(hydroxymethyl)aminomethane (TRIS).

5.2.13  Tris(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl).

5.2.14  CTAB extraction buffer solution, containing:

— CTAB (5.2.4), mass concentration ρ = 20 g/l,

— sodium chloride (5.2.10), molar concentration c = 1,4 mol/l,

— TRIS (5.2.12), c = 0,1 mol/l,

— Na₂EDTA (5.2.3), c = 0,02 mol/l.

The pH is adjusted to 8,0 by adding hydrochloric acid (5.2.5).

5.2.15  Proteinase K solution, ρ = 20 mg/ml.

The freshly produced Proteinase K solution should be stored in the form of aliquots at −20 °C.

5.2.16  Amylase solution, ρ = 10 mg/ml.

Weigh 100 mg of α-amylase (5.2.9) into a sterile glass container and add 10 ml of double distilled, sterile water.

The solution should be kept refrigerated after preparation until analysis. Frozen storage is possible, whereby repeated freezing and thawing should be avoided.

Any sediment in the solution has no negative influence on the test.

5.2.17  Solution buffer for RNase A, containing:

— Tris-HCl (5.2.13) (c = 0,01 mol/l);

— sodium chloride (5.2.10) (c = 0,015 mol/l).

The pH is adjusted to 7,5 with hydrochloric acid (5.2.5) or sodium hydroxide solution (5.2.11).

5.2.18  RNase A solution, ρ = 10 mg/ml.

Weigh 100 mg RNAse A (5.2.8) into a sterile glass jar and add 10 ml RNase A solution buffer (5.2.17). Heat for 15 min to 100 °C and cool down to room temperature slowly.

The aliquoted solution can be stored frozen at -20 °C for at least 12 months.

5.2.19  CTAB precipitation buffer solution, containing:

— CTAB (5.2.4) (ρ = 5 g/l);

— sodium chloride (5.2.10) (c = 0,04 mol/l).

5.2.20  Sodium chloride solution, c = 1,2 mol/l.

5.2.21  0,2 × TE buffer solution, containing:

— TRIS (5.2.12), c = 0,002 mol/l,

— Na₂EDTA (5.2.3), c = 0,000 2 mol/l.

The pH is adjusted to 8,0 by adding hydrochloric acid (5.2.5) or sodium hydroxide solution (5.2.11).

NOTE     For all solutions commercially available alternatives can be used.

5.3 Real-time PCR reagents

5.3.1 Master mix for real-time PCR, containing thermostable DNA polymerase (for hot-start PCR) and PCR buffer solution (containing reaction buffer, dNTPs, MgCl₂), as a dilutable concentrate.

Ready to use reagents or single components may be used as a PCR master mix.

5.3.2 Oligonucleotides

The oligonucleotides are shown in Table 1.

Table 1 — Primers and probes for the real-time PCR

6 Apparatus and equipment

6.1 General

In addition to the typical laboratory facilities, the following equipment shall be used.

Due to the high sensitivity of PCR analytics and the risk for DNA contaminations, the use of aerosol-protected filter tips in the DNA extraction procedure is mandatory. Plastic and glass materials shall be sterilized and free of DNA before use.

Further general requirements are given in EN ISO 21571 [1].

6.2 DNA extraction

6.2.1  Suitable reaction vials, 1,5 ml and 2 ml, DNA-free.

6.2.2  50 ml centrifuge tubes, sterile.

6.2.3  Thermostat or water bath, preferably with shaker function.

6.2.4  Centrifuge, suitable for centrifuging 50 ml centrifuge tubes at 6 000 g.

If no centrifuge with a correspondingly high g-force is available, it is also possible to centrifuge at a speed of about 4 000 g^2[2]​ for a correspondingly extended time until a supernatant is obtained.

6.2.5  Centrifuge, suitable for centrifuging 1,5 ml and 2 ml reaction vials at 14 500 g.

6.2.6  Equipment and/or material for grinding the sample, e.g. blender or mill.

6.2.7  Equipment for DNA quantity estimation (optional), e.g. UV-photometer.

6.2.8  Vacuum dryer (optional).

6.2.9  Mechanical quick shaker, e.g. vortex mixer.

6.3 PCR

6.3.1  Suitable PCR tubes.

6.3.2  Microcentrifuge for PCR tubes.

6.3.3  Real-time PCR equipment, suitable for excitation and for emission measurement of fluorescence-marked oligonucleotides.

7 Procedure

7.1 General

General aspects are described in EN 15634-1 [5]and EN ISO 21571 [1].

7.2 Sample preparation

It should be ensured that the test sample taken after milling or homogenizing is representative of the laboratory sample.

7.3 Preparation of DNA extracts

7.3.1 DNA extraction with CTAB and DNA purification

The sample DNA analysed in the ring trial were obtained using the DNA extraction method described below.

The use of a commercially available kit is acceptable in place of the DNA extraction procedure described below, provided it yields comparable or better results.

In parallel to the test samples, the controls listed in 7.4.5 and 7.4.6 should be performed adequately.

The analyses should be carried out twice in accordance with the following scheme:

— Weigh 2 g of the homogenized sample into a 50 ml centrifuge tube (tube A).

— Add 10 ml of CTAB extraction buffer solution (5.2.14) and 20 μl of amylase solution (5.2.16). Suspend well by rotating the centrifuge tube around the longitudinal axis. Incubate for 30 min at 65 °C while stirring or gently shaking; alternatively, use a hybridization oven

— Add 20 µl of Proteinase K solution (5.2.15) and mix.

— Incubate and shake for 90 min at 65 °C and mix.

— Centrifuge for 10 min at 6 000 g at room temperature.

— Place 900 µl of chloroform (5.2.1) in a 2 ml reaction vial (tube B).

— Add 900 µl of supernatant from tube A to tube B and mix thoroughly for 30 s.

— Centrifuge for 10 min at about 14 500 g at room temperature for phase separation.

— The interphase should be as small as possible, if necessary, mix briefly again and centrifuge for another 20 min.

— Add 600-650 µl of supernatant from tube B to a new 2 ml reaction vial (tube C).

— Add 2 parts by volume of CTAB precipitation buffer solution (5.2.19) and mix.

— Incubate for 60 min at room temperature.

— Centrifuge for 10 min at about 14 500 g at room temperature.

— Carefully remove and discard the supernatant.

— Incubate the precipitate in 900 μl sodium chloride solution (5.2.20) for at least 30 min at 37 °C while gently shaking to dissolve the pellet.

— Add 10 μl RNase A solution (5.2.18) and incubate for 10 min at 37 °C.

— Add 900 μl chloroform and mix for 30 s (vortex mixer).

— Centrifuge for 10 min at 14 500 g at room temperature.

— Add the supernatant (the upper, aqueous phase, approx. 800 µl) from tube C to a new 1,5 ml reaction vial (tube D).

— Add 0,6 parts by volume (approx. 480 μl) of isopropyl alcohol (5.2.6) and mix carefully by turning upside down several times.

— Incubate at room temperature for at least 20 min (if necessary, overnight).

— Centrifuge for 10 min at 14 500 g at room temperature.

— Carefully remove and discard the supernatant.

— Wash pellet with 500 μl ethanol (70 %) (5.2.2).

— Centrifuge for 10 min at 14 500 g at room temperature.

— Carefully pour off the supernatant completely and discard it.

— Dry extracted DNA at room temperature or under vacuum.

— Dissolve dried DNA in 50 μl 0,2 × TE buffer (5.2.21): agitate gently for 1 hour at 37 °C or for several hours at room temperature, possibly keeping it refrigerated overnight.

The DNA extracts can be stored refrigerated for a short period or should be stored frozen. Repeated freezing and thawing should be avoided.

7.3.2 Optional quantification of DNA concentration

The concentration of a DNA aliquot can be determined by UV spectrophotometry at a wavelength of 260 nm with the following Formula (1):

In order to check its purity, the sample may in addition be measured at 280 nm. The ratio of the values for optical density at wavelengths of 260 nm and 280 nm should be approximately 1,8.

The DNA mass concentration may also be estimated using other suitable procedures.

NOTE     The DNA concentration determined by this method is influenced by the matrix and the processing state of the extracted sample. The DNA concentration determined this way does not allow any conclusion on the content of nut DNA in the sample.

7.4 Real-time PCR set-up

7.4.1 Reaction mix for real-time PCR

As an example, the procedure is described below for a total reaction volume of 25 µl (comprising 20 µl of PCR mix and 5 µl of DNA extract), using the reagents listed in Table 2. The final reagent concentrations shown in Table 2 have been proven suitable. The PCR can also be performed with different volumes, provided the reagent concentrations are adjusted accordingly.

Prior to use, reagents should be gently thawed, for example on ice or a cooling block, and briefly centrifuged. During PCR mix preparation, reagents should be kept on ice or in a cooling block, if necessary. Care should be taken to thoroughly mix each reagent immediately before pipetting.

A PCR mix should be prepared containing all components except the DNA extract. The required volume of PCR mix should be calculated based on the number of reactions to be performed, including a 10 % safety margin. Each DNA extract should be analysed in duplicate. For each reaction, 5 µl of DNA extract should be added. Alongside the test samples, the controls specified in 7.4.2 to 7.4.6 should be included.

Table 2 — Reaction mix for real-time PCR

— Mix the PCR reagents (see Table 2), centrifuge shortly and pipette 20 µl per PCR in each reaction vial.

— For each of the sample reactions, pipette 5 µl of sample DNA extract into the PCR master mix.

— For the positive control for DNA targets (7.4.2), pipette 5 µl of the target containing DNA into the PCR master mix.

— For the negative control for DNA targets (7.4.3), pipette 5 µl of the peanut free sample DNA extract into the PCR master mix.

— For the amplification reagent control (7.4.4), pipette 5 µl of water into the PCR master mix.

— For the extraction blank control (7.4.5), pipette 5 µl of extract from the negative extraction control sample into the PCR master mix.

— For the positive extraction control (7.4.6), pipette 5 µl of the peanut-containing sample DNA into the PCR master mix.

— Place the reaction vials in the PCR device and start the temperature/time program.

7.4.2 Positive control for DNA targets

The positive control is a control reaction containing the target DNA in a specified quantity or number of copies. DNA for the positive PCR control is extracted from pure nut samples,, as described in 7.3.1.

7.4.3 Negative control for DNA targets

The negative control is a sample of the food matrix without target sequence, which passes through all steps of the analytical process.

7.4.4 Amplification reagent control

The amplification reagent control (No template control) is a control containing all reagents, except extracted test sample template DNA. Instead of the template DNA, a corresponding volume of nucleic acid free water or buffer is added to the reaction.

7.4.5 Extraction blank control

The extraction blank control is a control performing all steps of the DNA extraction procedure, except addition of the test portion, e.g. by substitution of a corresponding amount of water for the test portion.

7.4.6 Positive extraction control

The positive extraction control is a sample of the food matrix with known quantity of nuts, which passes through all steps of the analytical process, e.g. rice biscuits containing 400 mg peanut, hazelnut, walnut and cashew per kg food.

7.4.7 Temperature/time program (real-time PCR)

The temperature/time program indicated in Table 3 has proven effective for reaction vials made from plastic for the present PCR.

Table 3 — Temperature/time program for plastic reaction vessels

7.4.8 Accept/Reject criteria

The results obtained, including those from the controls, should be unambiguous and match the expected outcomes based on the control reactions. If this is not the case, the entire procedure—from DNA extraction onward—should be repeated. The results from the two test sample replicates should be consistent. If one replicate yields a positive result and the other a negative result, the analysis shall be repeated. If feasible, the amount of template nucleic acid in the reaction should be increased to ensure consistent results across both replicates. Data evaluation should be performed using the appropriate analysis software specific to the PCR device. The indication of amplification may vary depending on the real-time PCR system used. In the case of a negative result (no detectable PCR product), the output may display terms such as “undetermined,” “no amp,” or the maximum number of cycles run. For a positive result (amplification of the target DNA sequence), the cycle during which the fluorescence from the reaction crosses a specified threshold level at which the signal can be distinguished from background levels —the Ct (cycle threshold) or Cp (crossing point) value—should be determined.

If the automatic evaluation fails to provide a valid result due to atypical fluorescence data, manual adjustment of the baseline and threshold may be required. In such cases, the device-specific instructions provided in the software manual should be followed.

The PCR result shall be reported as:

— Positive, if a specific PCR product is detected above the LOD and all controls yield expected results; or

— Negative, if no specific PCR product is detected above the LOD and all controls yield expected results.

7.4.9 Identification

Verification of a positive detection is obtained by the sequence-specific hybridization of the real-time hydrolysis probe.

8 Validation

8.1 General

The method described in this document was elaborated by the working group “Lebensmittelallergene“ (Food Allergens) of the Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (Federal Office of Consumer Protection and Food Safety, BVL) pursuant to § 64 of the German Food and Feed Code (LFGB) and validated in a ring trial with a total of 12 participants. Prior to the ring trial it was validated in a single laboratory by the method developers [4].

8.2 Specificity

Prior to the ring trial (see 8.4), the specificity was randomly tested in one individual laboratory under the conditions chosen for the ring trial.

No cross reactivity was observed with:

For Cashew, additional species from the Sumac group were tested: Peruvian and Brazilian pepper tree (Schinus molle and Schinus terebinthifolia).

For Hazelnut, the following species were additionally tested: Alfalfa, Buckwheat, Chickpea, Coconut, and Quinoa. For hazelnut, a strong amplification signal with the non-food specie birch was detected.

No crosstalk was observed when testing 100 ng of each of the four target species per PCR assay.

Furthermore, the variation of the response was tested when analyzing materials of the target species from different origins and – if available- of different cultivars. For the calculation of the response, the concentration of ng nut DNA/µl was determined using an external standard curve with the spiked nut material as reference (= 100 %) for all materials. The response values were calculated as the ratio between the concentration of nut DNA of the analysed nut material and the “reference” nut material. In Table 4, the mean values of all results and the relative standard deviation in relation to the mean (CV %) are given.

Table 4 — Evaluation of response variation

A sufficiently low variation of the response was observed for all detection systems ranging from 17 % to 37 % (Table 4).

The detection method for hazelnut is only genus-specific (Corylus spp.). The test of the quantitative response was carried out with commercially available references, predominantly derived from the Lambert's nut (C. maxima). Equally, nuts from the common hazel (C. avellana) are detected by this method. The results of the tests with available references hint to a response of C. avellana being about factor two to four stronger than the response of C. maxima [4].

8.3 Sensitivity

The sensitivity of the method was tested using a rice biscuit matrix spiked with 400 mg/kg of each of the four target species (using flour from each respective species) (see 8.4). A dilution of the DNA extract from this matrix control, corresponding to 1 mg/kg per species, remained detectable in all 6 out of 6 reactions for each case.

In a pre-ring trial involving five laboratories, a freshly prepared DNA dilution corresponding to 0,64 mg/kg of each nut was detected consistently across all laboratories for all four species, with successful amplification in all 42 reactions.

Additionally, the limit of detection (LOD) was assessed under asymmetric conditions using the rice biscuit sample spiked at 5 mg/kg (see also 8.4). These concentrations remained detectable in all 6 out of 6 reactions, even after the addition of 250 ng of DNA from each of the three non-target species.

8.4 Method validating interlaboratory study (ring trial)

8.4.1 Setup of the ring trial

The reliability of the method was evaluated in a ring trial involving 12 participating laboratories [4]. Materials of different food matrices (rice biscuits, cooked sausage, sauce powder, vegan cookie and veggie burger (powder) were artificially contaminated (incurred) with defatted flours of peanut, hazelnut, cashew and walnut (hereafter referred to as “nuts”) and subsequently processed [2] [6] [7]. Rice biscuits were baked at 200 °C for 10 min, sausage meat mixtures of the type “Lyoner” were filled into cans and boiled at 100 °C for 1,5 h. For sauce powder no further processing step was applied. Artificially contaminated vegan cookies and veggie burgers were provided by DLA Proficiency Testing (Oering, Germany)[7]. Only these two materials were spiked with roasted nuts.

In addition, a rice biscuit was used to prepare the matrix positive control DNA. Therefore, rice biscuits were spiked with defatted flours of peanut, hazelnut, walnut and cashew (each non-roasted) in proportions of 400 mg/kg each before baking [6]. The spiking materials were not identical. Only in the case of the rice biscuit (400 mg/kg) used as matrix positive control and rice biscuit 2, the identical nut flour was spiked. All materials were stored frozen until analysis.

An overview of the materials and the respective amounts of ground nuts (flour) is provided in Table 5. DNA was extracted centrally by the organising laboratory according to section 7.3.1 and aliquots were distributed to the ring trial participants.

Table 5 — Materials used for the ring trial

Each participating laboratory received 21 coded DNA solutions. These were derived from seven different materials, with three identical DNA solutions provided for each spiking level. In addition, each laboratory received pre-extracted DNA for preparing a dilution series of the positive extraction control, as specified in Table 6.

Participants were provided with the master mix^3[3]​, primers, and probes for real-time PCR.

There were no restrictions on the choice of PCR instrument. Each DNA sample was analysed in duplicates. Genomic DNA extracted from rice biscuits containing 400 mg/kg nut was further diluted with 0.2× TE buffer (5.2.21) shortly before PCR, as specified in Table 6. Levels 1 to 6 were analysed in triplicate, while level 7 served as a sensitivity control and was tested ten times.

Participants received a data sheet to record Ct values and calculate standard curve criteria using the positive control dilution series (Table 6 , level 1-6).

Table 6 — Dilution scheme of the positive extraction control

8.4.2 Deviations from the ring trial protocol

No deviations from the ring trial protocol were reported.

8.4.3 Ring trial validation results

8.4.3.1 Results for the positive control dilution series

Table 7 summarises the results for the matrix dilution series (see Table 6).

Table 7 — Ring-trial: Nuts DNA, real-time PCR devices and results of the dilution series

Due to the lack of appropriate guidelines and performance criteria related to allergen analysis, guidelines for real-time PCR based GMO analyses were used for evaluation of the results: According to the ENGL method acceptance criteria, the average value of the slope of the standard curve shall range from -3,1 to -3,6 and the coefficient of determination R2 should be ≥ 0,98 [8][9].

Amplification efficiency and R2 coefficient are not applicable to qualitative methods. Values outside the specifications do not automatically result in the exclusion of the laboratory in question. In the case of dilution series, however, they provide information about the quality of the series and the robustness of the method.

For laboratory no. 12, these criteria were clearly outside the expected range for all four nut species. The results of laboratory 12 were therefore removed from the data set as an outlier.

For the walnut system, two other laboratories (no 8 and 10) had significant deviations for both criteria. Therefore, the results of these two laboratories were removed for evaluation of the walnut PCR criteria.

Overall, the requirements for slope and coefficient of determination were met by 9–11 laboratories for each specie (see Table 7).

The lowest matrix dilution, corresponding to 0,64 mg of the respective "nut specie"/kg, underwent a 10‑fold examination each and served simultaneously as a sensitivity check. In total, positive reactions were obtained in 110 of 110 reactions (= 100 %) (laboratory 12 not considered, see above).

8.4.3.2 Results for the ring trial samples – qualitative evaluation

After assignment of the results to the seven different materials or their corresponding extracted DNA samples (see Table 5), six values were obtained for each material and laboratory (three sample preparations, each analysed in duplicate).

The qualitative evaluation of the ring trial study is summarised in Table 8, Table 9, Table 10 and Table 11.

Peanut

Table 8 — Qualitative evaluation of the ring trial study for peanut

For the evaluation of positive results, two approaches were applied. First, all reported amplifications with Ct values below 38 were considered. Second, an evaluation was conducted by comparing the Ct values of the samples with those reported by the respective laboratory for the lowest matrix dilution (0,64 mg/kg) [10].

For each of the four allergenic ingredients tested, the expected results were obtained, with some exceptions.

One of 66 PCR reactions for the DNA extract from rice biscuit 2 (0,9 mg/kg peanut) was excluded from analysis, as deviating results (false negatives or significantly elevated Ct values) were also observed for this sample in two other systems. Method-related deviations were therefore considered unlikely. In the case of rice biscuit 3 (no peanut added), one out of 66 reactions showed amplification with a Ct value of 37,9. When comparing this result with the average Ct value for the lowest matrix dilution (0,64 mg/kg) reported by the corresponding laboratory, it was not classified as a false positive.

For all four spiked allergenic ingredients, consistent positive reactions were obtained when considering reported amplifications with Ct < 38. However, when evaluating the vegan cookie sample (5 mg/kg peanut) by Ct comparison with the lowest matrix dilution, 29 out of 66 reactions (44 %) were classified as false negatives. This may be due to the use of roasted peanuts for spiking, which significantly reduces detection sensitivity—by more than one order of magnitude [4]. In contrast, the veggie burger (powder) sample (10 mg/kg peanut), also prepared with roasted peanuts, consistently yielded positive results with both evaluation methods.

For rice biscuit 1 (5 mg/kg peanut), a single false-negative result was observed under the Ct comparison approach, representing 1,5% of all reactions. These findings demonstrate that reliable detection of 5 mg/kg peanut—equivalent to approximately 1,3 mg peanut-derived protein per kg (according to the Federal Food Code, BLS)—is achievable in processed foods. In cases where roasted peanuts are used instead of unroasted ones, a limit of detection (LOD) of 10 mg/kg can still be attained.

Hazelnut

Table 9 — Qualitative evaluation of the ring trial study for hazelnut

For rice biscuit 3 (no hazelnut added), amplification with Ct values below 38 was observed in 15 of 66 reactions. The reported Ct values were 34,4 or higher, with most falling in the range of 37 to 38. When comparing these Ct values with those reported for the lowest concentration level of the dilution series (0,64 mg/kg) [10], none of the results were classified as false positives. A minor contamination of the rice biscuit material, which served as the “zero standard”, is suspected.

For the other unspiked sample, six false-positive reactions were recorded under the Ct < 38 evaluation criterion. However, all six results originated from a single laboratory. The veggie burger (powder) material had not been specifically prepared as a hazelnut-free “zero standard”, and was therefore designated with a nominal spiking level of "0".

For the spiked materials, consistent positive responses were obtained for all matrices and reactions using both evaluation approaches, including the vegan cookie, which was prepared using roasted hazelnuts.

These findings confirm that hazelnut at 5 mg/kg—equivalent to approximately 0,7 mg hazelnut-derived protein per kg (according to the Federal Food Code, BLS)—can be reliably detected in processed foods, even when roasted hazelnuts are present.

Walnut

Table 10 — Qualitative evaluation of the ring trial study for walnut

For the veggie burger (powder) sample without walnut addition, amplification with Ct values below 38 was observed in 38 of 66 reactions. The reported Ct values were 34,3 or higher. When comparing these values with those of the lowest matrix dilution standard (0,64 mg/kg) [10], none of the results were classified as false positives. These findings confirm the indications from preliminary trials, which suggested walnut contamination of the frying powder at levels close to the detection limit. The material had not been specifically prepared as a walnut-free “zero standard” and was therefore designated with a nominal spiking level of "0".

For the two other unspiked samples, only one false-positive reaction was recorded under the Ct < 38 evaluation criterion. This supports the assumption that the false-positive results observed for the veggie burger (powder) were due to low-level contamination rather than method-related issues.

For all spiked materials, consistent positive results were obtained across all reactions and matrices using both evaluation approaches, including the vegan cookie prepared with roasted walnuts.

These results demonstrate that walnut at 5 mg/kg—equivalent to approximately 0,7 mg walnut-derived protein per kg (according to the Federal Food Code, BLS)—can be reliably detected in processed foods, even when roasted walnuts are used.

Cashew

Table 11 — Qualitative evaluation of the ring trial study for cashew

With the exception of one false-negative reaction each for rice biscuit 1 and the sauce powder (both containing 5 mg/kg cashew), observed under the Ct < 38 evaluation criterion, consistent positive results were obtained for all reactions and all spiked materials using both evaluation approaches—including the vegan cookie prepared with roasted cashew nuts.

These findings confirm that 5 mg/kg cashew, or even lower concentrations, can be reliably detected in processed foods. This corresponds to approximately 0,9 mg cashew-derived protein per kg (according to the Federal Food Code, BLS), and reliable detection remains achievable even when roasted cashew is used.

9 Test report

The test report should comply with EN ISO/IEC 17025 [11] and contain at least the following information:

a) all information necessary for the identification of the sample (kind of sample, origin of sample, designation);

b) a reference to this document, including its year of publication;

c) the date and type of sampling procedure (if known);

d) the date of receipt;

e) the date of test;

f) the test results according to EN 15634-1 [12];

g) any particular points observed in the course of the test;

h) any operations not specified in the method or regarded as optional, which might have affected the results.

Bibliography

Bibliography

1. ​¹  NCBI-GenBank® is an example of a suitable search tool for free use. This information is given for the convenience of users of this document and does not constitute an endorsement by CEN. ↑

2. ​²  g = 9,81 m⋅s^–2 ↑

3. ​³  QuantiTect® Multiplex Mastermix no ROX, QIAGEN GmbH, Hilden was used in the ring trial and constitutes an example of a suitable product commercially available. This information does not constitute an endorsement by CEN of this product. ↑