gmo - screening qualitative reports 2015/report dla... · august 2015 dla – 24/2015 –...

August 2015 DLA – 24/2015 – GMO-Screening qualitative

DLADienstleistung

LebensmittelAnalytik GbR

Evaluation Reportproficiency test

24/2015

GMO - Screening qualitative:

5 Samples with positive/negative amounts of GMO-Maize (Bt11) or GMO-Soya (RR)

Dienstleistung Lebensmittel Analytik GbRWaldemar-Bonsels-Weg 17022926 Ahrensburg, Germany

[email protected] www.dla-lvu.de

Coordinator of this PT: Dr. Matthias Besler

Reprint, also in part, only with written permission from DLA-Ahrensburg of 25


Content1. Introduction..................................................32. Realisation...................................................3

2.1 Test material............................................32.2 Test.....................................................32.3 Submission of results....................................3

3. Evaluation....................................................54. Results.......................................................5

4.1 Test.....................................................64.1.1 Results: 35S-Screening-Sequence........................64.1.2 Results: NOS-Screening-Sequence........................74.1.3 Results: GMO-Soya (RR-Round-Up-Ready-Soya).............84.1.4 Results: Lectin-DNA (Soya-specific)....................94.1.5 Results: GMO-Maize (bt11-Maize).......................104.1.6 Results: Maize-DNA (Maize-specific)...................114.1.7 Results: Other Parameters (DNA).......................12

5. Documentation................................................135.1 Details by participants about DNA-Extraction methods. . . .135.1.1 35S-Screening Sequence................................135.1.2 NOS-Screening Sequence................................145.1.3 GMO-Soya (RR-Round-Up-Ready-Soya).....................155.1.4 Lectin-DNA (Soya-specific)............................155.1.5 GMO-Maize (bt11-Maize)................................165.1.6 Maize-DNA (Maize-specific)............................165.1.7 Other Parameters (DNA)................................175.2 Details by participants to PCR-reaction.................185.2.1 35S-Screening Sequence................................185.2.2 NOS-Screening Sequence................................195.2.3 GMO-Soya (RR-Round-Up-Ready-Soya).....................205.2.4 Lectin-DNA (Soya-specific)............................215.2.5 GMO-Maize (bt11-Maize)................................225.2.6 Maize-DNA (Maize-specific)............................225.2.7 Other Parameters (DNA)................................23

6. Index of participant laboratories............................247. Index of references..........................................25



1. Introduction

The participation in proficiency testing schemes is an essential elementof the quality-management-system of every laboratory testing food andfeed, cosmetics and food contact materials. The implementation ofproficiency tests enables the participating laboratories to prove theirown analytical competence under realistic conditions. At the same timethey receive valuable data regarding the validity of the particulartesting method. The purpose of DLA is to offer proficiency tests for selected parametersin concentrations with practical relevance.Realisation and evaluation of the present proficiency test follows thetechnical requirements of DIN EN ISO/IEC 17043 (2010) and DIN ISO13528:2009.

2. Realisation

2.1 Test material

The test materials are 5 different mixtures of common in commerce foodsfrom European and US-American suppliers (s. table 1).The ingredients were mixed, homogenized and portioned to approximately10 g. The materials were tested for homogeneity.

2.2 Test

One portion of each of the 5 test materials was sent to every participa-ting laboratory in the 21st week of 2015. The testing method was optio-nal. The tests should be finished at July 3rd 2015 the latest.

2.3 Submission of results

The participants submitted their results in standard forms, which havebeen handed out along with the samples. The results given aspositive/negative were evaluated with respect to each tested parameter.Queried and documented were the indicated results and details of the testmethods like specifity, test kit manufacturer and hints about the pro-cedure.All participants submitted their results in time.


http://dict.leo.org/ende?lp=ende&p=/gQPU.&search=retrieval

http://dict.leo.org/ende?lp=ende&p=/gQPU.&search=retrieval


Table 1: Composition of DLA-samples

DLA-Sample

Ingredients (per 100 g) GMO-Con-tent Maize

GMO-Con-tent Soya

1 Maize Semolina, European-Supplier (62,5 g)Ingredients: Maize FlourNutrients per 100 g: Protein 7,5 g, Carbohydrates 74 g, Fat 1 g

Potato Flour (37,5 g)Ingredients: Potato flourIngredients per 100 g: Protein 0,6 g, Carbohydrates 83 g, Fat 0,1 g

-

-

-

-

2 Maize Semolina, European-Supplier (50 g)Ingredients: Maize FlourNutrients per 100 g: Protein 7,5 g, Carbohydrates 74 g, Fat 1 g


Maize Flour, USA-Supplier (12,5 g)Ingredients: Maize FlourNutrients per 100 g: Protein 9 g, Carbohydrates 79 g, Fat 0 g

-

-

positive(bt11-Maize experimental)

-

-

-

3 Soy Flakes, European Supplier (62 g)Ingredients: SoybeansNutrients per 100 g: Protein 41 g, Carbohydrates 3,1 g, Fatt 21 g

Glucose (19 g)Lactose (19 g)

-

- -

-

- -

4 Meal Replacement, Dietetic Food (62,5 g)Ingredients: Soyprotein isolate, honey, skimmed milk – yoghurt powder, iron-fumarate, zinc oxide, copper gluconate, manganese sulfate, potassium iodide, vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, biotin, calcium pantothenate, folic acid, nicotinamide, vitamin D3, dl-alpha-toco-pheryl acetate


-

-

positive(RR-Soya experimental)

-

5 Soy Flakes, European Supplier (59 g)Ingredients: SoybeansNutrients per 100 g: Protein 41 g, Carbohydrates 3,1 g, Fatt 21 g

Glucose (18 g)Lactose (18 g)

Soya Chunks, USA-Supplier (5,4 g)Ingredients: Soybean FlourNutrients per 100 g: Protein 47 g, Carbohydrates 17 g, Fat 0,8 g

-

- -

-

-

- -

positive(RR-Soya experimental)



3. Evaluation

The evaluation of the GMO-screening proficiency test was done exclusivelyqualitative.

The results are presented for all 5 test samples in separate tables foreach parameter 35S, NOS, GMO-Soya (RR), Lectin-DNA, GMO-Maize (bt11),Maize-DNA and other DNA results.The numbers and percentage of positive and negative results are given atthe end of each table. The participating laboratories decide according totheir own criteria wether a result is valuated positive or negative.If there are ≥ 75 % positive or negative results, a consensus result isdetermined for each sample.

For every participant a qualitative valuation is made with respect to theconsensus results. Therefore the number and percentage of "correct"results of consensus results is given.

4. Results

All following tables are anonymized. With the delivering of the evalua-tion-report the participants are informed about their individual evalua-tion-number.

The results of the participants are given in tables as indicated below:


Sample 1 Sample 2 Sample 3 Sample 4 Sample 4

Parameter

Evaluation number

Qualitative Valuation

Remarks

pos/neg pos/neg pos/neg pos/neg pos/neg Agreements with consensus results


4.1 Test

4.1.1 Results: 35S-Screening-Sequence

Comments on results:

For 4 samples consensus results with two times 100%, one time 90% and onetime 86% positive or negative results were obtained.For sample 3 there were 71% negative and 29% positive results. So aconsensus value with ≥ 75% could not be determined. Therefore sample 3was not considered for the "qualitative valuation".



35S

1 positive positive negative positive positive 3/4 (75%)

2 negative positive positive positive positive 4/4 (100%)

3 negative positive negative positive positive 4/4 (100%)

4 positive positive positive positive positive 3/4 (75%)










15 negative positive negative negative positive 3/4 (75%)








Sample 1 Sample 2 Sample 3 Sample 4 Sample 53 21 6 19 2118 0 15 2 014 100 29 90 10086 0 71 10 0

negative positive positive positive

Evaluation number


Remarks

pos/neg pos/neg pos/neg pos/neg pos/negAgreements with consensus value

Sample 3: tracesbelow

absolute LOD

Sample 3: traces ct 38,5

Number positive

Number negative

Percent positive

Percent negative

Consensus none


4.1.2 Results: NOS-Screening-Sequence


For all 5 samples consensus results with two times 100% and one time 90%,86% and 81% each positive or negative results were obtained.



NOS























negative positive negative positive positive

Evaluation number


Remarks

pos/neg pos/neg pos/neg pos/neg pos/negAgreements with con-

sensus value

Sample 3: traces below absolute

LOD

* Sample 3: traces ct 38,8

Number positive

Number negative

Percent positive

Percent negative

Consensus


4.1.3 Results: GMO-Soya (RR-Round-Up-Ready-Soya)


For 4 samples consensus results with one time 100%, two times 86% and onetime 79% positive or negative results were obtained.For sample 4 there were 71% positive and 29% negative results. So aconsensus value with ≥ 75% could not be determined. Therefore sample 4was not considered for the "qualitative valuation".



1 positive negative positive positive positive 2/4 (50%)

2 negative negative negative negative positive 4/4 (100%)

3 negative negative negative positive positive 4/4 (100%)

5 negative negative positive positive positive 3/4 (75%)












negative negative negative positive

Evaluation number


Remarks

RR-Soya pos/neg pos/neg pos/neg pos/neg pos/negAgreements with con-

sensus value

Sample 3: traces below relative LOD

Number positive

Number negative

Percent positive

Percent negative

Consensus none


4.1.4 Results: Lectin-DNA (Soya-specific)


For 3 samples consensus results with 100% each positive or negativeresults were obtained.For samples 1 and 2 there were 67% negative results. Therefore noconsensus value could be determined and the samples were not included forthe "qualitative valuation".



Lectin












21 positive negative positive positive positive 3/3 (100%)


positive positive positive

Evaluation number


Remarks

pos/neg pos/neg pos/neg pos/neg pos/neg Agreements with consensus value

Sample 1 + 2 late amplification curve, not

clearly positive

Sample 1: few traces

Number positive

Number negative

Percent positive

Percent negative

Consensus none none


4.1.5 Results: GMO-Maize (bt11-Maize)


For all 5 samples consensus results with three times 100% and two times90% positive or negative results were obtained.



1 negative positive negative positive negative 4/5 (80%)

3 negative positive negative negative negative 5/5 (100%)









Sample 1 Sample 2 Sample 3 Sample 4 Sample 50 10 0 1 110 0 10 9 9

Prozent positive 0 100 0 10 10Prozent negative 100 0 100 90 90

negative positive negative negative negative

Evaluation number


Remarks

bt11 Maize pos/neg pos/neg pos/neg pos/neg pos/neg Agreements with consensus value

Number positive

Number negative

Consensus


4.1.6 Results: Maize-DNA (Maize-specific)


For 3 samples consensus results with two times 92% and one time 83%positive results were obtained.For samples 3 and 5 there were 50% positive and 50% negative results.Therefore no consensus value of ≥ 75% could determined for samples 3 and5 and they were not included for the "qualitative valuation". Accordingto participants' remarks only „traces“ of maize DNA were detected insamples 3 and 5.



1 positive positive positive positive positive 3/3 (100%)3 positive positive negative positive positive 3/3 (100%)5 positive positive positive positive positive 3/3 (100%)6 positive positive negative negative negative 2/3 (100%)


12 positive positive negative positive negative 3/3 (100%)

13 positive positive positive positive positive 3/3 (100%)15 negative negative negative negative negative 0/3 (0%)17 positive positive positive positive positive 3/3 (100%)18 positive positive negative positive negative 3/3 (100%)19 positive positive negative positive negative 3/3 (100%)

21 positive positive positive positive negative 3/3 (100%)


positive positive positive

Evaluation number


Remarks

Maize DNA pos/neg pos/neg pos/neg pos/neg pos/negAgreements with consen-

sus value

Sample 3 + 5: low content of Maize DNA

Sample 3 + 5: traces;Sample 4: clear traces

Samplen 3+4: low traces

Number positive

Number negative

Percent positive

Percent negative

Consensus none none


4.1.7 Results: Other Parameters (DNA)


Parameter Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

1a NK603 positive positive negative positive positive1b negative positive negative positive positive1c bar negative negative negative negative negative1d FMV negative negative negative negative negative1e EPSPS positive negative positive positive2 Chloroplast-DNA positive positive positive positive positive4 FMV negative negative negative negative positive5 FMV negative negative negative negative positive7 FMV negative negative negative negative positive8 negative negative negative negative negative

11 negative positive negative positive positive

12a p35S-pat negative positive negative negative negative12b p35S-pat negative positive negative negative negative

12c negative positive negative positive positive

12d bar negative negative negative negative negative12e negative negative negative negative negative14 FMV negative negative negative positive positive15 negative negative negative negative negative20 Plant-DNA positive positive positive positive positive

21a negative positive negative positive positive

21b negative positive negative positive positive

21c Screening bar negative negative negative negative negative21d Screening FMV negative negative negative negative positive

21e negative negative negative negative positive

24 FMV negative negative negative negative positive

Evaluation number

Remarks

Other DNA pos/neg pos/neg pos/neg pos/neg pos/neg

pat

pos / neg

other

CTP2-CP4-EPSPS

CTP2-CP4-EPSPS

Samples 4 + 5: clear traces

pNOS

Target-DNA

Screening Pat-Gen

Screening CTP2-CP4-EPSPS

GVO-Soja MON89788

Sample 4: traces


5. Documentation

5.1 Details by participants about DNA-Extraction methods

5.1.1 35S-Screening Sequence


35S

12

3 §64 LFGB

45

6

7

8

9

10

11 ASU L 00.00-122

12 p35S-spezifische DNA-Sequenzen ASU L 00.00-122

14 35S1516 35 S, NOS

18 P-35S

1920 ASU L00.00.31

21 35S-promoter

22 GEN-IAL24 35S

Evaluation number

Result given as Test-Kit or Literature Remarks to DNA-Extraction

Target-Sequenz / -DNA Supplier / Method e.g. Extraction / Enzymes / Clean-Up / DNA-Quality

extract. 2g with CTAB and clean-up with QIAamp® DNA Stool Mini Kit (Fa. Qiagen)

combination: CTAB-Lysis with Macherey Nagel NucleoSpin® Food Kit

35S CaMV-Promotor SureFood GMO Kit, R-Biopharm NucleoSpin Food, Fa. Machery & NagelGen-ial FFS-Kit, Promega

detected or not detected Target-Sequence 35S promotor

LLC “Spectrtrading” 1) Qualitative 35S/tNOS GMO Detection Kit;

1 step extraction with PrepMan ®ultra Sample Preparation Reagent proceid by Applied Biosystems by Life Technologies

Congen: Sure Food GMO/ Screen 35S/NOS/FMV

extraction with SureFood Prep Advaced

Biotecon Diagnostics

35S-Promoter DNA in FAM-Kanal SureFood® GMO Screen 4plex 35S/NOS/FMV+IAC

Extraction by SureFood® PREP DNA/RNA Virus

unknown Biotecon Biotecon foodproof Sample Prep. Kit III (S 400 06.1)Promotor des Cauliflower Mosaic

Virus (CaMV35S)Machery&Nagel NucleoSpin Food Kit / 200 mg sample weight

ASU § 64 LFGB L 15.05-1 (SDS/Guanidinium chloride-buffer with Proteinase K, clean-up by Wizard-Kit from Fa. Promega)

R-Biopharm R-Biopharm, SureFood PREP Advanced, according to manualTarget - DNA Biotecon Diagnostics foodproof GMO Sample Preparation Kit

Biotecon Biotecon DNA preapration kitfoodproof GMO Screening 1 LyoKit-5'Nuclease-, R 602 17-1, biotecon

DNA extraction according to foodproof Magnetic Preparation Kit III, biotecon S 400 13L

Macherey Nagel - nucleobondmodified CTAB-method with clean-up

Microsynth Pentaplex AllGVOscB

Macherey-Nagel Nucleospin Food Kit with own optimization; Proteinase K and RNase A; Clean-up via Silica columns; DNA-content and -quality photometric determination: sample 1: 48 ng/µl; A260/A280=1.8 A260/A230=2.1; sample 2: 48 ng/µl;

A260/A280=1,8 A260/A230=2,2 sample 3: 97ng/µl; A260/A280=1,9 A260/A230=1,5 sample 4: 146 ng/µl; A260/A280=1,9 A260/A230=1,6 sample 5: 67 ng/µl;

A260/A280=1,9 A260/A230=1,5

Congen 35S / nos / FMV Monoplex Congen PREP Basic extraction kit


5.1.2 NOS-Screening Sequence


NOS

1

2

3 §64 LFGB

45

6

7

8

9

10 unbekannt

11 ASU L 00.00-122

12 ASU L 00.00-122

14 NOS1516 35 S, NOS

18 T-NOS

1920 ASU L00.00.31

21

22 GEN-IAL24

Evaluation number



extract. 2g with CTAB and clean-up with innuPREP DNA Micro Kit (845-KS-1010050; Analytik Jena)

QIAamp® DNA Stool Mini Kit (Fa. Qiagen)combination: CTAB-Lysis with Macherey Nagel NucleoSpin® Food

KitNOS-Terminator SureFood GMO Kit, R-Biopharm NucleoSpin Food, Fa. Machery & Nagel

Gen-ial FFS-Kit, Promegadetected or not detected Target-

Sequence NOS terminator2) 35S soybean qualitative/quantitative

GMO Detection Kit; Quality assessment of DNA extract was not conducted

Congen: Sure Food GMO/ Screen 35S/NOS/FMV

Biotecon Diagnostics

NOS-Terminator DNA in Cy5-KanalSureFood® GMO Screen 4plex

35S/NOS/FMV+IACExtraction by SureFood® PREP DNA/RNA Virus

Biotecon Biotecon foodproof Sample Prep. Kit III (S 400 06.1)Terminator des Agrobacterium

tumefaciens (nos)Machery&Nagel NucleoSpin Food Kit / 200 mg sample weight

tNOS-spezifische DNA-SequenzenASU § 64 LFGB L 15.05-1 (SDS/Guanidinium chloride-buffer with

Proteinase K, clean-up by Wizard-Kit from Fa. Promega)R-Biopharm R-Biopharm, SureFood PREP Advanced, according to manual

Target - DNA Biotecon Diagnostics foodproof GMO Sample Preparation KitBiotecon Biotecon DNA preapration kit

foodproof GMO Screening 1 LyoKit-5'Nuclease-, R 602 17-1, biotecon

DNA extraction according to foodproof Magnetic Preparation Kit III, biotecon S 400 13L

Macherey Nagel - nucleobondmodified CTAB-method with clean-up

Nos-terminator Microsynth Pentaplex AllGVOscBMacherey-Nagel Nucleospin Food Kit with own optimizations;

Proteinase K and RNase A; Clean-up with Silica columns;

nos Congen 35S / nos / FMV Monoplex Congen PREP Basic Extraction kit


5.1.3 GMO-Soya (RR-Round-Up-Ready-Soya)

5.1.4 Lectin-DNA (Soya-specific)


1 L 00.00 1052

3 §64 LFGB

5

6

11

12

13 P35S/CTP1517

18

19

21 GTS 40-3-2

23

Evaluation number


RR-Soya Target-Sequenz / -DNA Supplier / Method e.g. Extraction / Enzymes / Clean-Up / DNA-Quality

see aboveQIAamp® DNA Stool Mini Kit (Fa. Qiagen)


Gen-ial FFS-Kit, Promegadetected or not detected Soya RR 40-3-2 event-specific sequence

3) 35S maize qualitative/quantitative GMO Detection Kit;

Machery&Nagel NucleoSpin Food Kit / 200 mg sample weightp35S-CTP1 Konstrukt spezifische

DNA-SequenzenASU L 00.00-105, Annex C2


ASU L 00.00-105 Annex C.2 CTAB, Proteinase K, RNase A + QIAquickTarget - DNA Biotecon Diagnostics foodproof GMO Sample Preparation Kit

Target-Sequence EURL-Method Nucleo-Spin Food Kit (Maccherey & Nagel)

RRs foodproof GMO RR Soya Quantification

Kit-5'Nuclease-, R 302 19, bioteconDNA extraction according to foodproof Magnetic Preparation Kit III,

biotecon S 400 13L

Macherey Nagel - nucleobond

Microsynth Pentaplex All SoyMacherey-Nagel Nucleospin Food Kit with own optimizations;

Proteinase K and RNase A; Clean-up with Silica columns; Sure Food

Lectin

1 L 00.00 1052

3 §64 LFGB

5

6

11 Lectin-Gene ASU L 00.00-105

12

13 Lectin-Gene17

18

19

21 Lectin

Evaluation number



see aboveQIAamp® DNA Stool Mini Kit (Fa. Qiagen)


Gen-ial FFS-Kit, Promegadetected or not detected taxon-specific sequence soy (fragment

lectin gen)

4) RR40-3-2 GMO line soybean qualitative Detection Kit;

Machery&Nagel NucleoSpin Food Kit / 200 mg sample weight

Lectin specific DNA-Sequences ASU L 00.00-105, Annex C2ASU § 64 LFGB L 15.05-1 (SDS/Guanidinium chloride-buffer with

Proteinase K, clean-up by Wizard-Kit from Fa. Promega)ASU L 00.00-105 Annex C.2 CTAB, Proteinase K, RNase A + QIAquick

Target-Sequence EURL-Methode Nucleo-Spin Food Kit (Maccherey & Nagel)

lectinfoodproof GMO RR Soya Quantification

Kit-5'Nuclease-, R 302 19, bioteconDNA extraction according to foodproof Magnetic Preparation Kit III,

biotecon S 400 13L


Microsynth Pentaplex AllGVOscBMacherey-Nagel Nucleospin Food Kit with own optimizations; Proteinase K and RNase A; Clean-up with Silica columns;


5.1.5 GMO-Maize (bt11-Maize)

5.1.6 Maize-DNA (Maize-specific)


bt11-Maize

1

3 §64 LFGB

5

6

13 ASU L 15.05-11517

18 Bt11

19

21 Bt-11

Evaluation number



see abovecombination: CTAB-Lysis with Macherey Nagel NucleoSpin® Food

KitGen-ial FFS-Kit, Promega

detected or not detected Maize bt11 event-specific sequence

5) bt11 GMO line maize qualitative Detection Kit

IVS2-2/PAT-B-Primer CTAB, Proteinase K, RNase A + QIAquickTarget - DNA Biotecon Diagnostics foodproof GMO Sample Preparation Kit

Target-Sequence EURL-Method Nucleo-Spin Food Kit (Maccherey & Nagel)SureFood GMO QUANT Bt11 Corn,

S2016, r-biopharmDNA extraction according to foodproof Magnetic Preparation Kit III,

biotecon S 400 13LMacherey Nagel - nucleobond

JRC protocol event-specific method for the quantification of maize event Bt11

Macherey-Nagel Nucleospin Food Kit with own optimizations; Proteinase K and RNase A; Clean-up with Silica columns;

1

3 §64 LFGB

5

6

11 ASU L 00.00-105

12

131517

18

19

21

Evaluation number



see abovecombination: CTAB-Lysis with Macherey Nagel NucleoSpin® Food

KitGen-ial FFS-Kit, Promega

detected or not detected taxon-specific sequence maize (fragment

zein gen)

Made from reagents manufactured by Applied Biosystems/ Life Technologies

hmg-Gen Machery&Nagel NucleoSpin Food Kit / 200 mg sample weight

Invertase specific DNA-SequencesBrodmann et al. (2002) J. of AOAC Int.

85(3): 646-653ASU § 64 LFGB L 15.05-1 (SDS/Guanidinium chloride-buffer with

Proteinase K, clean-up by Wizard-Kit from Fa. Promega)HMG-Gene ASU L 00.00-105 Anh. D.2 CTAB, Proteinase K, RNase A + QIAquick

Target - DNA Biotecon Diagnostics foodproof GMO Sample Preparation KitTarget-Sequence EURL-Method Nucleo-Spin Food Kit (Maccherey & Nagel)

zeinSureFood GMO QUANT Bt11 Corn,

S2016, r-biopharmDNA extraction according to foodproof Magnetic Preparation Kit III,

biotecon S 400 13L


mhmg Microsynth Pentaplex AllGVOscBMacherey-Nagel Nucleospin Food Kit with own optimizations; Proteinase K and RNase A; Clean-up with Silica columns;


5.1.7 Other Parameters (DNA)


1a

1b

1c

1d

1e24 Promotor578

11 ASU L 00.00-125

12a ASU G 30.40-1

12b

12c ASU L 00.00-125

12d

12e ASU L 00.00-141

14 FMV15

20

21a

21b CP2 & CP4 EPSPS

21c Bar-Gen

21d

21e MON89788

24 FMV

Evaluation number



see above [tables]

see above [tables]

see above [tables]

see above [tables]

see above [tables]QIAamp® DNA Stool Mini Kit (Fa. Qiagen)

SureFood GMO Kit, R-Biopharm NucleoSpin Food, Fa. Machery & NagelGen-ial FFS-Kit, Promega

Biotecon DiagnosticsTransition from CTP2 to CP4-

EPSPS-GeneMachery&Nagel NucleoSpin Food Kit / 200 mg sample weight

p35S-pat construct specific DNA-Sequence


p35S-pat construct specific DNA-sequences

Methodensammlung der Länderarbeitsgemeinschaft Gentechnik

(AM001)


CTP2-CP4-EPSPS construct specific DNA-sequences


bar-specific DNA-sequences ASU L 00.00-124, modifiedASU § 64 LFGB L 15.05-1 (SDS/Guanidinium chloride-buffer with

Proteinase K, clean-up by Wizard-Kit from Fa. Promega)

pNOS specific DNA-sequencesASU § 64 LFGB L 15.05-1 (SDS/Guanidinium chloride-buffer with

Proteinase K, clean-up by Wizard-Kit from Fa. Promega)R-Biopharm R-Biopharm, SureFood PREP Advanced, nach Anleitung

Target - DNA Biotecon Diagnostics foodproof GMO Sample Preparation KitScreening Plant-DNA / in-house-

methodmodifiziertes CTAB-Verfahren mit clean-up

Pat-Gen Microsynth Tetraplex AllGVOscCMacherey-Nagel Nucleospin Food Kit with own optimizations;


Microsynth Tetraplex AllGVOscCMacherey-Nagel Nucleospin Food Kit with own optimizations;


Microsynth Tetraplex AllGVOscCMacherey-Nagel Nucleospin Food Kit with own optimizations;


FMV promoter Microsynth Tetraplex AllGVOscCMacherey-Nagel Nucleospin Food Kit with own optimizations;


Microsynth Pentaplex All SoyMacherey-Nagel Nucleospin Food Kit with own optimizations;

Proteinase K and RNase A; Clean-up with Silica columns; Congen 35S / nos / FMV Monoplex Congen PREP Basic Extraction kit


5.2 Details by participants to PCR-reaction

5.2.1 35S-Screening Sequence


1 Real Time PCR2

3 Real Time PCR 4

5 Real Time PCR

6

7

8

9

10

11

12

14

15

16 Real time PCR18

19 real time PCR20

21

22

24

Evaluation number

Notes to PCR-Reaction Further Remarks

e.g. Real Time PCR / Gel electrophoresis / Cycles / Lenght of Amplif icates / Refe-rence material

Gel electrophoresis/ 45 Cycles/ 123 bp/ Bt11-Maize, RR-Soya tested in double determination

RT PCR, 45 Cycles

Real time PCR ( 7300 Real time PCR system). PCR protocol (amplification) : 1) stage 1 (initial steps: AmpEraseUNG activation) 2 min 50 °C - 1 cycles; 2) stage 2 (initial steps: Ampli Tag DNA polymerase

activation) 10 min 95 °C - 1 cycles; 3) stage 3 ( each of 40 cycles) denaturation 15 sec 95 °C, anneal/extend 1 min 58 °C

Sample 2: 35S in the range of LODReal Time PCR /Multiplex

Real Time PCR, 45 Cycles, internal Amplification controlreal-time PCR (Biotecon foodproof GMO Screening Kit (R 302 17))

Real-time PCR; 45 Cycles; 82 bp; Reference material gv-Maize Bt11Sample 3: traces below absolute LOD

(valuated negative)Real Time PCR, SureFood GVO 4plex, R-Biopharm, according to

manualfoodproof GMO Screenig Kit

real time PCR/ 50 cycles

Gel electrophoresisReal-Time PCR m. 45 Cycles; certified reference material: GTS 40-3-2 Soya (IRMM) with 0.1%, 1% and 10% GMO-content; for control of

inhibition sample eluates were tested in 1:5 and 1:25 dilutions; negative control (Nuclease-free H2O instead of DNA)

Real Time PCR, 45 Cycles, Reference material ERM-BF 410dk * Sample 3: traces ct 38,5


5.2.2 NOS-Screening Sequence


1 Real Time PCR2

3 Real Time PCR 4

5 Real Time PCR

6

7

8

9

10

11

12

14

15

16 Real time PCR18

19 real time PCR20

21

22

24

Evaluation number



Gel electrophoresis/ 45 Cycles/ 123 bp/ Bt11-Maize, RR-Soya tested in double determination

RT PCR, 45 Cycles

PCR controls: 1) PCR inhibition control - reaction mix with plasmid DNA ; 2) PCR reagent control (negative control) - reaction mix with nucleic acid free water; 3) PCR positive DNA target control (positive control) - reaction mix with DNA extact (10% RR 40-3-2 soya; 10 % bt11; 2% MON 810 test-

kit №3)

Real Time PCR /Multiplex

Real Time PCR, 45 Cycles, internal Amplification controlreal-time PCR (Biotecon foodproof GMO Screening Kit (R 302 17))

Real-time PCR; 45 Cycles; 84 bp; Reference material gv-Maize Bt11Sample 3: traces below absolute LOD

(valuated negative)Real Time PCR, SureFood GVO 4plex, R-Biopharm, according to

manualfoodproof GMO Screenig Kit


Gel electrophoresisReal-Time PCR m. 45 Cycles; certified reference material: GTS 40-3-2 Soya (IRMM) with 0.1%, 1% and 10% GMO-content; for control of


Real Time PCR, 45 Cycles, Reference material ERM-BF 410dk * Sample 3: traces ct 38,8


5.2.3 GMO-Soya (RR-Round-Up-Ready-Soya)


1 Real Time PCR

23 Real Time PCR

5 Real Time PCR

6

11

12

1315

17

1819 real time PCR

21

23 real time PCR

Evaluation number



Gel electrophoresis/ 45 Cycles/ 169 bp/ RR-Soya tested in double determination

Сertified reference material: Institute of Reference Materials and Measurements,Geel Belgium

Real-time PCR; 45 Cycles; 74 bp; Reference material gm-Soya GTS 40-3-2

Sample 3: traces below relative LOD (valuated negative)

Real-time PCR 74 bp

foodproof GMO Screenig Kit

Real Time PCR / 45 Cycles / Reference material JRC (Geel Belgium) /10 to 100 ng DNA per well


Real-Time PCR m. 45 Cycles; certified reference material: GTS 40-3-2 Soya (IRMM) with 0.1%, 1% and 10% GMO-content, as well as 0%

GMO-Soya. For identification 200 ng (40 ng/µl) DNA each was applied. Negative control (Nuclease-free H2O instead of DNA)

Sample 4: RR-content in total Soya <0,1%; Sample 5: RR-content 5,2%


5.2.4 Lectin-DNA (Soya-specific)


1 Real Time PCR

23 Real Time PCR

5 Real Time PCR

6

11

12

13

17

1819 real time PCR

21

Evaluation number



Gel electrophoresis/ 45 Cycles/ 438 bp/ RR-Soya tested in double determination

Roundup ReadyTM soya beans (ERM-BF410 ak; ERM-BF410 bk; ERM-BF410 dk; ERM-BF410 gk)

Real-time PCR; 45 Cycles; 74 bp; Reference material gm-Soya GTS 40-3-2

Real-time PCR 74 bpSamples 1 + 2 showed late amplification

curves, not clearly positiveReal Time PCR / 45 Cycles / Reference material JRC (Geel Belgium) /10

to 100 ng DNA per well


Real-Time PCR m. 45 Cycles; certified reference material: GTS 40-3-2 Soya (IRMM) with 0.1%, 1% and 10% GMO-content; for control of


Sample 1 low soya traces


5.2.5 GMO-Maize (bt11-Maize)

5.2.6 Maize-DNA (Maize-specific)


1 Real Time PCR

3 Real Time PCR

5 Real Time PCR

6

1315

17

1819 real time PCR

21

Evaluation number



MON 810 maize (ERM-BF413 ak; ERM-BF413 ck; ERM-BF413 ek; ERM-BF413 gk;)

PCR 189 bp + Gel electrophoresis + Restriction 116+73 bp


Real Time PCR / 45 cycles / Reference material JRC (Geel Belgium) /10 to 100 ng DNA per well


Real-Time PCR 45 cycles; certified reference material: Bt-11 Maize (IRMM) with 0.1%, 1% and 5% GMO-content; For identification 200 ng (40

ng/µl) DNA each were applied. Negative control (Nuclease-free H2O instead of DNA)

Sample 2: 0,9% Bt-11 in total Maize

1 Real Time PCR

3 Real Time PCR

5 Real Time PCR

6

11

12

1315

17

1819 real time PCR

21

Evaluation number



Bt-11 maize (ERM-BF412 f)

Samples 3 and 5 not suitable for analysis of gm-maize, they contain too less amplifyable maize DNA. Sample 4: sample specific LOD

of gm-maize 0,7 %.

Real-time PCR; 45 cycles; 79 bp; Reference material gm-maize Bt11

Samples 3 and 5: traces of maize (valuation negative);

sample 4: clear traces of maize (contamination?) detected (valuation

positive)

Real-time PCR, 79 bp


Real Time PCR / 45 cycles / Reference material JRC (Geel Belgium) /10 to 100 ng DNA per well


Real-Time PCR 45 cycles; for inhibition control sample eluates were 1:5 and 1:25 diluted

Samplles 3+4: low maize traces


5.2.7 Other Parameters (DNA)


1a Real Time PCR

1b Real Time PCR

1c Real Time PCR

1d Real Time PCR

1e Real Time PCR

245 Real Time PCR

78 bar-gen,/FMV

11

12a

12b

12c

12d12e141520

21a

21b

21c

21d

21e

24

Evaluation number



multiple extraction dif fering results, at LOD?

Gel electrophoresis/ 45 Cycles/ 200-600 bp/ Bt11-Maize, Bt176, RR-Soya tested in double determination

RT PCR, 45 Cycles

Real Time PCR /Multiplex

Real-time PCR; 45 cycles; 102/111 and 402 bp (Bt11) respectively; Reference material gm-Maize Bt11

conventional PCR-RFLP; 45 cycles; 370 and 550 bp (Bt11); Reference material gm-Maize Bt11 and T25

Sample 2: size of PCR-amplif icates and restriction fragments of positive sample 2 not in agreement

w ith gm-maize Bt11

Real-time PCR; 45 cycles; 88 bp; Reference material gm-rape Gt73Samples 4 and 5: clear traces (contamination?)

detected (valuated positive)

Real-time PCR; 45 cycles; 110 bp; reference material gm-rape Ms1xRf1

Real-time PCR; 45 cycles; 94 bp; reference material gm-rape Ms1xRf1

Real Time PCR, SureFood GVO 4plex, R-Biopharm, according to manual


Gel electrophoresis

Real-Time PCR 45 cycles; certif ied reference material: MON89788 Soya (IRMM) w ith 0.1%, 1% and 10% GMO-content. negative control (Nuclease-free H2O

instead of DNA)

Real-Time PCR 45 cycles; certif ied reference material: MON89788 Soya (IRMM) w ith 0.1%, 1% and 10% GMO-content. negative control (Nuclease-free H2O

instead of DNA)

Real-Time PCR 45 cycles; negative control (Nuclease-free H2O instead of DNA)

Real-Time PCR 45 cycles; negative control (Nuclease-free H2O instead of DNA)

Real-Time PCR 45 cycles; certif ied reference material: MON89788 Soya (IRMM) w ith 0.1%, 1% and 10% GMO-content, as w ell as 0% GMO-Soya. For identif ication 200 ng (40 ng/µl) DNA w ere applied. Negative control (Nuclease-f ree H2O instead

of DNA)

Sample 5: MON89788-content in total soya <0,1%

Real Time PCR, 45 Cycles, reference material ERM-BF 410dk * Sample 4: traces ct 38,2


6. Index of participant laboratories

[Die Adressdaten der Teilnehmer wurden für die allgemeine Veröffentli-chung des Auswerte-Berichts nicht angegeben.]

[The address data of the participants were deleted for publication of the evaluation report.]


FRANCE

AUSTRIABELARUSNETHERLANDS

Teilnehmer / Participants Ort / TownGermanyGermanyGermany

GermanyGermanyGermanyGermanyGermanyGermanyGermanyGermanyGermanyGermanyGermanyGermanyGermanyGermany

GermanyGermany


7. Index of references

1. DIN EN ISO/IEC 17043:2010; Konformitätsbewertung – Allgemeine Anforderungen an Eignungsprüfungen / Conformity assessment – General requirements for proficiency testing

2. Verordnung / Regulation 882/2004/EU; Verordnung über amtliche Kontrollen / Regulation on official controls

3. DIN EN ISO/IEC 17025:2005; Allgemeine Anforderungen an die Kompetenz von Prüf- und Kalibrierlaboratorien / General requirements for the competence of testing and calibration laboratories

4. Richtlinie / Directive 1993/99/EU; über zusätzliche Maßnahmen im Bereich der amtlichen Lebensmittelüberwachung / on additional measuresconcerning the official control of foodstuffs

5. ASU §64 LFGB : Planung und statistische Auswertung von Ringversuchen zur Methodenvalidierung

6. DIN ISO 13528:2009; Statistische Verfahren für Eignungsprüfungen durchRingversuche / Statistical methods for use in proficiency testing by interlaboratory comparisons

7. The International Harmonised Protocol for the Proficiency Testing of Ananlytical Laboratories ; J.AOAC Int., 76(4), 926 – 940 (1993)

8. The International Harmonised Protocol for the Proficiency Testing of Ananlytical Chemistry Laboratories ; Pure Appl Chem, 78, 145 – 196 (2006)

9. Evaluation of analytical methods used for regulation of food and drugs;W. Horwitz; Analytical Chemistry, 54, 67-76 (1982)

10.A Horwitz-like funktion describes precision in proficiency test; M. Thompson, P.J. Lowthian; Analyst, 120, 271-272 (1995)

11.Protocol for the design, conduct and interpretation of method performance studies; W. Horwitz; Pure & Applied Chemistry, 67, 331-343(1995)

12.Recent trends in inter-laboratory precision at ppb and sub-ppb concentrations in relation to fitness for purpose criteria in proficiency testing; M. Thompson; Analyst, 125, 385-386 (2000)

13.European Network of GMO Laboratories, Definition of Minimum Performance Requirements for Analytical Methods of GMO Testing, Version 25-01-2005

14.Powell J, Owen L, Reliability of food measurements: the application of proficiency testing to GMO analysis, Accred Qual. Assur. 7, 392-402(2002)

15.Thompson M, GMO Proficiency testing: Interpreting z-scores derived from log-transformed data, amc technical brief, No. 18 Dec 2004

16.Thompson M et al., Scoring in Genetically Modified Organism Proficiency Tests Based on Log-Transformed Results, J. AOAC Int., 89(1), 232-239 (2006)

17.Žel J et al., Calculation of Measurement Uncertainty in Quantitative Analysis of Genetically Modified Organisms Using Intermediate Precision - A Practical Approach, J. AOAC Int., 90(2), 582-586 (2007)


gmo - screening qualitative reports 2015/report dla... · august 2015 dla – 24/2015 –...

Documents