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Method Article
Assessment of cry1Ab transgene cassette in commercial Bt corn MON810: gene, event, construct and GMO specific concurrent characterization
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Narinder Singh
commented on 18 March, 2008
My name is Narinder and I am working for a biotech company in India in its GMo testing Unit.I happend to see your CORRESPONDENCE Article which is quite eye opener for all of us in the area of GMO testing. I have few question on LOD on GM events: 1) How many seeds you need to develop a protocol for LOD at 0.01 % for say cotton event cry 1Ac( Is it 1 GM seeds in 9999 seeds Non GM seeds or 1 GM seed in 10,000 non GM seeds)? 2) Do we need to check the lOD on all background parents used in backcrossing to get th hybrid after gene tranfer?( I mean LOD detection on T1, T2,T4 or just on the final product sayready for release Cry 1Ac) 3)What could be the sampling strategy here, how many gram of the test sample we need from a large sample and then how to calulate the respective copy no. in the event to define LOD at 0.01%?How many repetitions or sub samples we need to take to reach LOD at 0.01 % so that we reach desired GM copy nos to define LOD at 0.01 %? 4) Do we define 0.01 % LOD as 1 GM seeds in 10,000 Non GM seeds or we define it in terms of GM copy nos in a test sample? I would request you to please answer my queries and if you have a protocol to carry out such experiment to define LOD at 0.01 % and 0.1 % , I would request you to please share with me if it has no Intellectual property right associated with it.or any relevant research paper which can help us to develop a protocol on LOD at 0.01 % in the cotton event Cr1 Ac. hoping to hear from you soon in this regards With warm regards Narinder singh.PhD Functional Head bioagriculture Avesthagen Limited Upperground Floor Innovator Building Internal Tech park Whitefield raod Bangalore-560066 India ph No.0091-802841165 ext 5082 Mobile No.:0091-9886017739
D. N. Kachru
commented on 30 March, 2008
Dear Dr. Narinder: Please refer to your comment,the answer to you queries follows: Your queries - Q1) How many seeds you need to develop a protocol for LOD at 0.01 % for say cotton event cry1Ac (Is it 1 GM seeds in 9999 seeds Non GM seeds or 1 GM seed in 10,000 non GM seeds)? Ans. If the weight of all seeds is equal, then 1 GM seed and 9999 Non GM seeds, total 10,000 seeds will be corresponding to 0.01%. If the seeds weight are not equal, reference material could be prepared on the basis of weight. Please refer to CRM preparation procedure adopted by Fluka/sigma. However, please see the procedure proposed in the link: Laura B, Petra H, Simon K, Van den Eede G (2001) Review of GMO detection and quantification techniques. European Commission, Joint Research Centre, Institute for Health and Consumer Protection, Food Products and Consumer Goods Unit, Ispra. http://www.osservaogm.it/pdf/JRCReview.pdf At 95% probability level, based on binomial distribution, 3000 grains are recommended for 0.1% LOD. The expected probability of GMO copies in a sample of 1000 ng of DNA, at concentration of 0.005% (pl see table 6) would be 18 or less. 2) Do we need to check the LOD on all background parents used in backcrossing to get the hybrid after gene transfer? (I mean LOD detection on T1, T2, T4 or just on the final product say ready for release Cry1Ac). Ans. This depends on regulatory compliance criterion fixed by the official agencies. To us, logic suggests to test only the final product ready for release. 3) What could be the sampling strategy here, how many gram of the test sample we need from a large sample and then how to calculate the respective copy no. in the event to define LOD at 0.01%? How many repetitions or sub samples we need to take to reach LOD at 0.01 % so that we reach desired GM copy nos to define LOD at 0.01 %? Ans. Please refer to file mentioned above. You would find that at 95% confidence level, for 1% GM material, the number of grains needs to be 299 non-GM seed plus 01 GM seed. For desired LOD of 0.1%, the sample size should be 10 times higher (~3000 seeds). For gene copy calculation (Table 5) and for expected probability of GMO copies in a 1000ng DNA (Table 6, page 45 & 46). For additional details you may read our Ms. Chandra K. Singh, Abhishek Ojha, D.N. Kachru (2007). Detection and Characterization of cry1Ac Transgene Construct in Bt-Cotton: Multiple Polymerase Chain Reaction Approach. J. AOAC International, 90 (6):1517-1525 http://www.atypon-link.com/AOAC/doi/abs/10.5555/jaoi.90.6.1517 4) Do we define 0.01% LOD as 1 GM seeds in 10,000 Non GM seeds or we define it in terms of GM copy nos in a test sample? Ans. For regulatory inspection, no. of GM seeds in a given lot would determine as LOD%. We only wish that one day copy no. is accepted as the ultimate proof for LOD. For a scientific research lab, copy no. makes absolute sense. I would request you to please answer my queries and if you have a protocol to carry out such experiment to define LOD at 0.01 % and 0.1 %, I would request you to please share with me if it has no Intellectual property right associated with it. or any relevant research paper which can help us to develop a protocol on LOD at 0.01 % in the cotton event Cry1Ac. Ans. Please see our another Ms — Chandra K. Singh, Abhishek Ojha, Raj K. Bhatnagar & D.N. Kachru (2008). Detection & characterization of recombinant DNA expressing vip3A type insecticidal gene in GMOs – Standard single, Multiplex & Construct specific PCR assays. Analytical & Bioanalytical Chemistry 2008 Jan; 390(1):377-387. http://www.springerlink.com/content/r5380436m41366v8/fulltext.pdf Hope the information provided in the link and our 02 Ms provide you the desired information. Thanks! Dr. D. N. Kachru
Narinder Singh
commented on 18 March, 2008
My name is Narinder and I am working for a biotech company in India in its GMo testing Unit.I happend to see your CORRESPONDENCE Article which is quite eye opener for all of us in the area of GMO testing. I have few question on LOD on GM events: 1) How many seeds you need to develop a protocol for LOD at 0.01 % for say cotton event cry 1Ac( Is it 1 GM seeds in 9999 seeds Non GM seeds or 1 GM seed in 10,000 non GM seeds)? 2) Do we need to check the lOD on all background parents used in backcrossing to get th hybrid after gene tranfer?( I mean LOD detection on T1, T2,T4 or just on the final product sayready for release Cry 1Ac) 3)What could be the sampling strategy here, how many gram of the test sample we need from a large sample and then how to calulate the respective copy no. in the event to define LOD at 0.01%?How many repetitions or sub samples we need to take to reach LOD at 0.01 % so that we reach desired GM copy nos to define LOD at 0.01 %? 4) Do we define 0.01 % LOD as 1 GM seeds in 10,000 Non GM seeds or we define it in terms of GM copy nos in a test sample? I would request you to please answer my queries and if you have a protocol to carry out such experiment to define LOD at 0.01 % and 0.1 % , I would request you to please share with me if it has no Intellectual property right associated with it.or any relevant research paper which can help us to develop a protocol on LOD at 0.01 % in the cotton event Cr1 Ac. hoping to hear from you soon in this regards With warm regards Narinder singh.PhD Functional Head bioagriculture Avesthagen Limited Upperground Floor Innovator Building Internal Tech park Whitefield raod Bangalore-560066 India ph No.0091-802841165 ext 5082 Mobile No.:0091-9886017739
D. N. Kachru
commented on 30 March, 2008
Dear Dr. Narinder: Please refer to your comment,the answer to you queries follows: Your queries - Q1) How many seeds you need to develop a protocol for LOD at 0.01 % for say cotton event cry1Ac (Is it 1 GM seeds in 9999 seeds Non GM seeds or 1 GM seed in 10,000 non GM seeds)? Ans. If the weight of all seeds is equal, then 1 GM seed and 9999 Non GM seeds, total 10,000 seeds will be corresponding to 0.01%. If the seeds weight are not equal, reference material could be prepared on the basis of weight. Please refer to CRM preparation procedure adopted by Fluka/sigma. However, please see the procedure proposed in the link: Laura B, Petra H, Simon K, Van den Eede G (2001) Review of GMO detection and quantification techniques. European Commission, Joint Research Centre, Institute for Health and Consumer Protection, Food Products and Consumer Goods Unit, Ispra. http://www.osservaogm.it/pdf/JRCReview.pdf At 95% probability level, based on binomial distribution, 3000 grains are recommended for 0.1% LOD. The expected probability of GMO copies in a sample of 1000 ng of DNA, at concentration of 0.005% (pl see table 6) would be 18 or less. 2) Do we need to check the LOD on all background parents used in backcrossing to get the hybrid after gene transfer? (I mean LOD detection on T1, T2, T4 or just on the final product say ready for release Cry1Ac). Ans. This depends on regulatory compliance criterion fixed by the official agencies. To us, logic suggests to test only the final product ready for release. 3) What could be the sampling strategy here, how many gram of the test sample we need from a large sample and then how to calculate the respective copy no. in the event to define LOD at 0.01%? How many repetitions or sub samples we need to take to reach LOD at 0.01 % so that we reach desired GM copy nos to define LOD at 0.01 %? Ans. Please refer to file mentioned above. You would find that at 95% confidence level, for 1% GM material, the number of grains needs to be 299 non-GM seed plus 01 GM seed. For desired LOD of 0.1%, the sample size should be 10 times higher (~3000 seeds). For gene copy calculation (Table 5) and for expected probability of GMO copies in a 1000ng DNA (Table 6, page 45 & 46). For additional details you may read our Ms. Chandra K. Singh, Abhishek Ojha, D.N. Kachru (2007). Detection and Characterization of cry1Ac Transgene Construct in Bt-Cotton: Multiple Polymerase Chain Reaction Approach. J. AOAC International, 90 (6):1517-1525 http://www.atypon-link.com/AOAC/doi/abs/10.5555/jaoi.90.6.1517 4) Do we define 0.01% LOD as 1 GM seeds in 10,000 Non GM seeds or we define it in terms of GM copy nos in a test sample? Ans. For regulatory inspection, no. of GM seeds in a given lot would determine as LOD%. We only wish that one day copy no. is accepted as the ultimate proof for LOD. For a scientific research lab, copy no. makes absolute sense. I would request you to please answer my queries and if you have a protocol to carry out such experiment to define LOD at 0.01 % and 0.1 %, I would request you to please share with me if it has no Intellectual property right associated with it. or any relevant research paper which can help us to develop a protocol on LOD at 0.01 % in the cotton event Cry1Ac. Ans. Please see our another Ms — Chandra K. Singh, Abhishek Ojha, Raj K. Bhatnagar & D.N. Kachru (2008). Detection & characterization of recombinant DNA expressing vip3A type insecticidal gene in GMOs – Standard single, Multiplex & Construct specific PCR assays. Analytical & Bioanalytical Chemistry 2008 Jan; 390(1):377-387. http://www.springerlink.com/content/r5380436m41366v8/fulltext.pdf Hope the information provided in the link and our 02 Ms provide you the desired information. Thanks! Dr. D. N. Kachru
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Method Article
Assessment of cry1Ab transgene cassette in commercial Bt corn MON810: gene, event, construct and GMO specific concurrent characterization
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
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CURRENT STATUS: Posted
Version 1
Posted 23 Oct, 2007
Community comments: 4
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Comments: 4
HTML Views: ...
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Narinder Singh
commented on 18 March, 2008
My name is Narinder and I am working for a biotech company in India in its GMo testing Unit.I happend to see your CORRESPONDENCE Article which is quite eye opener for all of us in the area of GMO testing. I have few question on LOD on GM events: 1) How many seeds you need to develop a protocol for LOD at 0.01 % for say cotton event cry 1Ac( Is it 1 GM seeds in 9999 seeds Non GM seeds or 1 GM seed in 10,000 non GM seeds)? 2) Do we need to check the lOD on all background parents used in backcrossing to get th hybrid after gene tranfer?( I mean LOD detection on T1, T2,T4 or just on the final product sayready for release Cry 1Ac) 3)What could be the sampling strategy here, how many gram of the test sample we need from a large sample and then how to calulate the respective copy no. in the event to define LOD at 0.01%?How many repetitions or sub samples we need to take to reach LOD at 0.01 % so that we reach desired GM copy nos to define LOD at 0.01 %? 4) Do we define 0.01 % LOD as 1 GM seeds in 10,000 Non GM seeds or we define it in terms of GM copy nos in a test sample? I would request you to please answer my queries and if you have a protocol to carry out such experiment to define LOD at 0.01 % and 0.1 % , I would request you to please share with me if it has no Intellectual property right associated with it.or any relevant research paper which can help us to develop a protocol on LOD at 0.01 % in the cotton event Cr1 Ac. hoping to hear from you soon in this regards With warm regards Narinder singh.PhD Functional Head bioagriculture Avesthagen Limited Upperground Floor Innovator Building Internal Tech park Whitefield raod Bangalore-560066 India ph No.0091-802841165 ext 5082 Mobile No.:0091-9886017739
D. N. Kachru
commented on 30 March, 2008
Dear Dr. Narinder: Please refer to your comment,the answer to you queries follows: Your queries - Q1) How many seeds you need to develop a protocol for LOD at 0.01 % for say cotton event cry1Ac (Is it 1 GM seeds in 9999 seeds Non GM seeds or 1 GM seed in 10,000 non GM seeds)? Ans. If the weight of all seeds is equal, then 1 GM seed and 9999 Non GM seeds, total 10,000 seeds will be corresponding to 0.01%. If the seeds weight are not equal, reference material could be prepared on the basis of weight. Please refer to CRM preparation procedure adopted by Fluka/sigma. However, please see the procedure proposed in the link: Laura B, Petra H, Simon K, Van den Eede G (2001) Review of GMO detection and quantification techniques. European Commission, Joint Research Centre, Institute for Health and Consumer Protection, Food Products and Consumer Goods Unit, Ispra. http://www.osservaogm.it/pdf/JRCReview.pdf At 95% probability level, based on binomial distribution, 3000 grains are recommended for 0.1% LOD. The expected probability of GMO copies in a sample of 1000 ng of DNA, at concentration of 0.005% (pl see table 6) would be 18 or less. 2) Do we need to check the LOD on all background parents used in backcrossing to get the hybrid after gene transfer? (I mean LOD detection on T1, T2, T4 or just on the final product say ready for release Cry1Ac). Ans. This depends on regulatory compliance criterion fixed by the official agencies. To us, logic suggests to test only the final product ready for release. 3) What could be the sampling strategy here, how many gram of the test sample we need from a large sample and then how to calculate the respective copy no. in the event to define LOD at 0.01%? How many repetitions or sub samples we need to take to reach LOD at 0.01 % so that we reach desired GM copy nos to define LOD at 0.01 %? Ans. Please refer to file mentioned above. You would find that at 95% confidence level, for 1% GM material, the number of grains needs to be 299 non-GM seed plus 01 GM seed. For desired LOD of 0.1%, the sample size should be 10 times higher (~3000 seeds). For gene copy calculation (Table 5) and for expected probability of GMO copies in a 1000ng DNA (Table 6, page 45 & 46). For additional details you may read our Ms. Chandra K. Singh, Abhishek Ojha, D.N. Kachru (2007). Detection and Characterization of cry1Ac Transgene Construct in Bt-Cotton: Multiple Polymerase Chain Reaction Approach. J. AOAC International, 90 (6):1517-1525 http://www.atypon-link.com/AOAC/doi/abs/10.5555/jaoi.90.6.1517 4) Do we define 0.01% LOD as 1 GM seeds in 10,000 Non GM seeds or we define it in terms of GM copy nos in a test sample? Ans. For regulatory inspection, no. of GM seeds in a given lot would determine as LOD%. We only wish that one day copy no. is accepted as the ultimate proof for LOD. For a scientific research lab, copy no. makes absolute sense. I would request you to please answer my queries and if you have a protocol to carry out such experiment to define LOD at 0.01 % and 0.1 %, I would request you to please share with me if it has no Intellectual property right associated with it. or any relevant research paper which can help us to develop a protocol on LOD at 0.01 % in the cotton event Cry1Ac. Ans. Please see our another Ms — Chandra K. Singh, Abhishek Ojha, Raj K. Bhatnagar & D.N. Kachru (2008). Detection & characterization of recombinant DNA expressing vip3A type insecticidal gene in GMOs – Standard single, Multiplex & Construct specific PCR assays. Analytical & Bioanalytical Chemistry 2008 Jan; 390(1):377-387. http://www.springerlink.com/content/r5380436m41366v8/fulltext.pdf Hope the information provided in the link and our 02 Ms provide you the desired information. Thanks! Dr. D. N. Kachru
Narinder Singh
commented on 18 March, 2008
My name is Narinder and I am working for a biotech company in India in its GMo testing Unit.I happend to see your CORRESPONDENCE Article which is quite eye opener for all of us in the area of GMO testing. I have few question on LOD on GM events: 1) How many seeds you need to develop a protocol for LOD at 0.01 % for say cotton event cry 1Ac( Is it 1 GM seeds in 9999 seeds Non GM seeds or 1 GM seed in 10,000 non GM seeds)? 2) Do we need to check the lOD on all background parents used in backcrossing to get th hybrid after gene tranfer?( I mean LOD detection on T1, T2,T4 or just on the final product sayready for release Cry 1Ac) 3)What could be the sampling strategy here, how many gram of the test sample we need from a large sample and then how to calulate the respective copy no. in the event to define LOD at 0.01%?How many repetitions or sub samples we need to take to reach LOD at 0.01 % so that we reach desired GM copy nos to define LOD at 0.01 %? 4) Do we define 0.01 % LOD as 1 GM seeds in 10,000 Non GM seeds or we define it in terms of GM copy nos in a test sample? I would request you to please answer my queries and if you have a protocol to carry out such experiment to define LOD at 0.01 % and 0.1 % , I would request you to please share with me if it has no Intellectual property right associated with it.or any relevant research paper which can help us to develop a protocol on LOD at 0.01 % in the cotton event Cr1 Ac. hoping to hear from you soon in this regards With warm regards Narinder singh.PhD Functional Head bioagriculture Avesthagen Limited Upperground Floor Innovator Building Internal Tech park Whitefield raod Bangalore-560066 India ph No.0091-802841165 ext 5082 Mobile No.:0091-9886017739
D. N. Kachru
commented on 30 March, 2008
Dear Dr. Narinder: Please refer to your comment,the answer to you queries follows: Your queries - Q1) How many seeds you need to develop a protocol for LOD at 0.01 % for say cotton event cry1Ac (Is it 1 GM seeds in 9999 seeds Non GM seeds or 1 GM seed in 10,000 non GM seeds)? Ans. If the weight of all seeds is equal, then 1 GM seed and 9999 Non GM seeds, total 10,000 seeds will be corresponding to 0.01%. If the seeds weight are not equal, reference material could be prepared on the basis of weight. Please refer to CRM preparation procedure adopted by Fluka/sigma. However, please see the procedure proposed in the link: Laura B, Petra H, Simon K, Van den Eede G (2001) Review of GMO detection and quantification techniques. European Commission, Joint Research Centre, Institute for Health and Consumer Protection, Food Products and Consumer Goods Unit, Ispra. http://www.osservaogm.it/pdf/JRCReview.pdf At 95% probability level, based on binomial distribution, 3000 grains are recommended for 0.1% LOD. The expected probability of GMO copies in a sample of 1000 ng of DNA, at concentration of 0.005% (pl see table 6) would be 18 or less. 2) Do we need to check the LOD on all background parents used in backcrossing to get the hybrid after gene transfer? (I mean LOD detection on T1, T2, T4 or just on the final product say ready for release Cry1Ac). Ans. This depends on regulatory compliance criterion fixed by the official agencies. To us, logic suggests to test only the final product ready for release. 3) What could be the sampling strategy here, how many gram of the test sample we need from a large sample and then how to calculate the respective copy no. in the event to define LOD at 0.01%? How many repetitions or sub samples we need to take to reach LOD at 0.01 % so that we reach desired GM copy nos to define LOD at 0.01 %? Ans. Please refer to file mentioned above. You would find that at 95% confidence level, for 1% GM material, the number of grains needs to be 299 non-GM seed plus 01 GM seed. For desired LOD of 0.1%, the sample size should be 10 times higher (~3000 seeds). For gene copy calculation (Table 5) and for expected probability of GMO copies in a 1000ng DNA (Table 6, page 45 & 46). For additional details you may read our Ms. Chandra K. Singh, Abhishek Ojha, D.N. Kachru (2007). Detection and Characterization of cry1Ac Transgene Construct in Bt-Cotton: Multiple Polymerase Chain Reaction Approach. J. AOAC International, 90 (6):1517-1525 http://www.atypon-link.com/AOAC/doi/abs/10.5555/jaoi.90.6.1517 4) Do we define 0.01% LOD as 1 GM seeds in 10,000 Non GM seeds or we define it in terms of GM copy nos in a test sample? Ans. For regulatory inspection, no. of GM seeds in a given lot would determine as LOD%. We only wish that one day copy no. is accepted as the ultimate proof for LOD. For a scientific research lab, copy no. makes absolute sense. I would request you to please answer my queries and if you have a protocol to carry out such experiment to define LOD at 0.01 % and 0.1 %, I would request you to please share with me if it has no Intellectual property right associated with it. or any relevant research paper which can help us to develop a protocol on LOD at 0.01 % in the cotton event Cry1Ac. Ans. Please see our another Ms — Chandra K. Singh, Abhishek Ojha, Raj K. Bhatnagar & D.N. Kachru (2008). Detection & characterization of recombinant DNA expressing vip3A type insecticidal gene in GMOs – Standard single, Multiplex & Construct specific PCR assays. Analytical & Bioanalytical Chemistry 2008 Jan; 390(1):377-387. http://www.springerlink.com/content/r5380436m41366v8/fulltext.pdf Hope the information provided in the link and our 02 Ms provide you the desired information. Thanks! Dr. D. N. Kachru