prEN 15634-7
Secretariat: CEN/TC 275
Foodstuffs - Detection of food allergens by molecular biological methods - Part 7: Peanut (Arachis hypogaea) - Qualitative detection of a specific DNA sequence in food by real-time PCR
Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen Verfahren - Teil 7: Erdnuss (Arachis hypogaea) - Qualitativer Nachweis einer spezifischen DNA-Sequenz in Lebensmitteln mittels Real-time PCR
CCMC will prepare and attach the official title page
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, and MgCl2), as a dilutable concentrate. 7
5.3.2 Oligonucleotides 7
6 Apparatus and equipment 7
6.1 General 7
6.2 DNA extraction 8
6.2.7 Equipment for DNA quantity estimation (optional), e.g. UV-photometer. 8
6.3 PCR 8
7 Procedure 8
7.1 General 8
7.2 Sample preparation 8
7.3 Preparation of DNA extracts 8
7.3.1 DNA extraction with CTAB and DNA purification 8
7.3.2 Optional quantification of DNA concentration 10
7.4 Real-time PCR set-up 10
7.4.1 Reaction mix for real-time PCR 10
7.4.2 Positive control for DNA targets 10
7.4.3 Negative control for DNA targets 10
7.4.4 Amplification reagent control 10
7.4.5 Extraction blank control 10
7.4.6 Positive extraction control 10
7.4.7 Temperature/time program (real-time PCR) 10
7.4.8 Accept/Reject criteria 10
7.4.9 Identification 10
8 Validation 10
8.1 General 10
8.2 Specificity 10
8.3 Sensitivity 10
8.4 Method validating interlaboratory study (ring trial) 10
8.4.1 Setup of the ring trial 10
8.4.2 Deviations from the ring trial study protocol 10
8.4.3 Ring trial validation results 10
9 Test report 10
Bibliography 10
This document (prEN 15634-7:2026) 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.
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.
This document specifies a method for the qualitative detection of peanut (Arachis hypogaea) DNA in food using real-time PCR and targeting a multicopy mitochondrial sequence, 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 in rice biscuits, wheat biscuits, cooked sausage and milk powder.
The limit of detection of the peanut real-time PCR has been determined experimentally to be at least 0,5 mg peanut/kg.
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
For the purposes of this document, the terms and definitions given in EN 15634‑1 and 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/
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. Real-time PCR is used to detect a 104 base pair (bp) long sequence targeting a multicopy sequence from mitochondrial peanut DNA close to the coding region of the ATPase subunit 6 (atp6) (Genbank1[1]: MW448460. The real-time PCR method involves a fluorescence detection with sequence-specific hydrolysis probes, see [2].
The following general conditions for analysis should be followed, unless specified differently. 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 relevant reagents in water and autoclaving, unless otherwise specified.
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 %.
5.2.9 α-Amylase (>1 500 U/mg Protein).
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), substance concentration c = 1,4 mol/l,
— TRIS (5.2.12), substance concentration c = 0,1 mol/l,
— Na2EDTA (5.2.3), substance concentration 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 is to 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 by adding 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 slowly to room temperature.
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.
5.3.1 Master mix for real-time PCR, containing thermostable DNA polymerase (for hot-start PCR) and PCR buffer solution (containing reaction buffer, dNTPs, and MgCl2), as a dilutable concentrate.
Ready to use reagents or single components may be used as a master mix.
An overview of the oligonucleotides is provided in Table 1 .
Table 1 — Primers and probes for the real-time PCR
DNA sequence of the oligonucleotide | |
|---|---|
5′ – FAM – AAg gCg AAg AAg ggT CAg AT – BHQ1 – 3′a | |
In addition to the typical laboratory facilities, the following equipment shall be used.
Due to the high sensitivity of the PCR analytics and the risk of 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].
6.2.1 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 g2[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.
6.3.2 Microcentrifuge for PCR tubes.
6.3.3 Real-time PCR equipment, suitable for excitation and for emission measurement of fluorescence-marked oligonucleotides.
General aspects are described in EN 15634-1 [3]and EN ISO 21571 [1].
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.
— Weigh 2 g of the homogenized sample into a 50 ml centrifuge tube (tube A).
— Add 10 ml of CTAB extraction buffer (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 Proteinase K solution (5.2.15) and mix.
— Incubate and shake for a further 90 min at 65 °C.
— 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 NaCl 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, if necessary 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):
(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 peanut DNA in the sample.
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
Final concentration | Volume per reaction [µl] | |
|---|---|---|
Forward Primer atp6-F (10 µmola) | ||
Reverse Primer atp6-R (10 µmola) | ||
TaqMan™ probe atp6-P (10 µmola) | ||
a Other working concentrations can be used. The volumes shall then be adjusted accordingly. | ||
— Mix the PCR master 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 peanuts, 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 peanut, which passes through all steps of the analytical process, e.g. rice biscuits containing 400 mg peanut 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
Parameter | Temperature | Time | Fluorescence measurement | Cycles | ||
|---|---|---|---|---|---|---|
15 mina | ||||||
a Follow the manufacturer's instructions of the PCR master mix. | ||||||
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.
Verification of a positive detection is obtained by the sequence-specific hybridization of the real-time hydrolysis probe.
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 the ring trial study with a total of 14 participants [4]. Prior to the ring trial it was validated in a single laboratory by the method developers [2].
The specificity of the method is documented in literature [2].
Prior to the ring trial (see 8.3), the specificity was randomly tested in one individual laboratory under the conditions chosen for the ring trial.
No cross reactivity was observed with:
Only cashew, Brazil nut and emmer showed a slight cross reaction of less than 0,01 %. However, it cannot be ruled out that the small signals are due to contamination of the material by peanut, since these were market samples.
In addition, various peanut varieties (unroasted) and commodities of roasted and unroasted peanuts were tested under the conditions chosen for the ring trial to confirm the literature data on variety- and processing-dependent variation [2]. The overall variation (relative standard deviation) of the response was below 50 % [4].
The sensitivity of the method was tested using a rice biscuit matrix spiked with 400 mg peanut per kg food (see 8.4). A dilution of the DNA extract from this matrix control, corresponding to 0,5 mg/kg, remained detectable in all 140 out of 140 reactions.
8.4 Method validating interlaboratory study (ring trial)
The reliability of the method was tested in a ring trial with a total of 14 participating laboratories [4]. The sample materials used were rice biscuits, wheat biscuits, cooked sausage and milk powder, each of which contained defined proportions of peanut [2][5][6]. The spiking levels for rice biscuits were: unspiked (blank), 5,3 mg/kg and 20,8 mg/kg. Wheat biscuits were spiked at levels of 0,5 mg/kg and 5 mg/kg; milk powder and cooked sausage with 5 mg/kg, respectively. Furthermore, rice biscuits were used to prepare the matrix positive control DNA, which was spiked with 400 mg/kg of peanut [5].
An overview of the sample materials used in the ring trial is provided in Table 4.
Table 4 — Materials used for the ring trial
Spiking (mg/kg) peanut | |
|---|---|
rice biscuit 400 a | |
DNA was extracted centrally by the organising laboratory according to section 7.3.1, and aliquots were distributed to the participating laboratories.
Each participating laboratory received 21 coded DNA solutions. These originated from seven different spiking levels or materials, with three identical solutions provided for each level. In addition, each laboratory received pre-extracted DNA for preparing a dilution series of the positive extraction control, as specified in Table 5.
Participants were provided with the master mix3[3], primers, and probes for real-time PCR.
There were no restrictions on the choice of PCR instrument. Each sample was analysed in duplicates. Genomic DNA extracted from rice biscuits containing 400 mg/kg peanut was further diluted with 0,2× TE buffer (5.2.21) shortly before PCR, as specified in Table 5. Levels 1 to 5 were analysed in triplicate, while level 6 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 5, level 1-5).
Table 5 — Dilution scheme of the positive control
Dilution procedure | Milligrams of peanut/kg | |
|---|---|---|
8.4.2 Deviations from the ring trial study 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 6 summarises the results for the matrix dilution series (see Table 5).
Table 6 — Ring trial: Peanut DNA, real-time PCR devices and results of the dilution series
Real-time PCR devicea | Slope | R2 | No. of positive rxn of the 0,5 mg/kg dilution | |
|---|---|---|---|---|
No. of positive amplifications / (percentage of positive amplifications) | ||||
a: Rotor-Gene™ Q (QIAGEN, Hilden) b: Applied Biosystems™ 7500 or 7500 Fast a Applied Biosystems™ 7500 or 7500 Fast, AGILENT™ Mx 3005P, Corbett Rotor-Gene™ 6000, Bio-rad CFX96™, Qiagen Rotor-Gene™ Q, AGILENT™ AriaMx, Roche LightCycler® 480 and Thermo Fisher QuantStudio are examples of suitable products available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by CEN of these products. | ||||
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 [7].
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.
All laboratories met both criteria. Each laboratory measured the lowest dilution corresponding to 0,5 mg peanut/kg in 10 replicates. In total, all 140 reactions yielded positive results (100 %).
One laboratory reported consistent results by testing a separate set of samples using a different commercial master mix for real-time PCR.
8.4.3.2 Results for the ring trial samples – qualitative evaluation
After assigning the results to the seven different materials or their extracted DNA samples (3 × rice biscuits, 2 × wheat biscuits, 1 × milk powder, 1 × cooked sausage), six PCR results were obtained for each material and laboratory (three DNA samples, each analysed in duplicate).
The qualitative evaluation of the ring trial study is summarised in Table 7 .
Table 7 — Qualitative evaluation of the ring trial study for peanut
Rice biscuits 0 | Rice biscuits 20,8 | Rice biscuits 5,3 | Wheat biscuits 0,5 | Wheat biscuits 5 | Milk powder 5 | Cooked sausage 5 | |
|---|---|---|---|---|---|---|---|
8a | |||||||
False-negative results (%)a | |||||||
a All positive amplifications considered, Ct < 45. b Evaluation by comparison of the Ct value with the average Ct value of the lowest matrix dilution (0,5 mg/kg) of the positive control material. If the Ct value (sample) was at least 1 higher than the Ct (0,5 mg/kg), then the result was assessed as negative[8]. For the samples which have no indicated evaluation methods a and b, no false-positive or false-negative results were obtained, regardless of the considered cut-off value. | |||||||
For the blank rice biscuit sample (0 mg peanut/kg), 8 out of 84 reactions showed false-positive amplification with Cq values below 45. However, in all cases, the corresponding replicate in the PCR duplicate was negative, leading to an overall evaluation of the duplicates as “negative”. The observed Cq values ranged from 38 to 41. Accordingly, no false-positive results were recorded when comparing the Cq values to those reported by the respective laboratory for the lowest positive control dilution (0,5 mg peanut/kg)[8].
When evaluating the wheat biscuit samples incurred with 0,5 mg peanut/kg [5] using the same approach, 40 out of 84 reactions were classified as false negatives (48%). This may be due to the fact that 0,5 mg peanut/kg corresponds to the lowest dilution level of the positive control DNA. According to this evaluation method, any Cq value at least one unit higher than the mean Cq of the lowest standard (0,5 mg peanut/kg) is interpreted as negative. However, when considering any detectable amplification regardless of Cq value, all PCR reactions at this level yielded positive results.
These findings confirm the assumed limit of detection (LOD) of 0,5 mg/kg for peanut detection.
The test report should comply with EN ISO/IEC 17025 [9] 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);
f) the test results according to EN 15634-1 [10];
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
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 g = 9,81 m⋅s–2 ↑
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. ↑
