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Wei J.,Shanghai JiaoTong University | Li F.,Jilin Agricultural University | Guo J.,Shanghai JiaoTong University | Li X.,GMO Detection Laboratory | And 4 more authors.
Journal of Agricultural and Food Chemistry | Year: 2013

The papaya (Carica papaya L.) Chymopapain (CHY) gene has been reported as a suitable endogenous reference gene for genetically modified (GM) papaya detection in previous studies. Herein, we further validated the use of the CHY gene and its qualitative and quantitative polymerase chain reaction (PCR) assays through an interlaboratory collaborative ring trial. A total of 12 laboratories working on detection of genetically modified organisms participated in the ring trial and returned test results. Statistical analysis of the returned results confirmed the species specificity, low heterogeneity, and single-copy number of the CHY gene among different papaya varieties. The limit of detection of the CHY qualitative PCR assay was 0.1%, while the limit of quantification of the quantitative PCR assay was ∼25 copies of haploid papaya genome with acceptable PCR efficiency and linearity. The differences between the tested and true values of papaya content in 10 blind samples ranged from 0.84 to 6.58%. These results indicated that the CHY gene was suitable as an endogenous reference gene for the identification and quantification of GM papaya. © 2013 American Chemical Society.


Li X.,GMO Detection Laboratory | Pan L.,GMO Detection Laboratory | Li J.,East China University of Science and Technology | Zhang Q.,East China University of Science and Technology | And 3 more authors.
Journal of Agricultural and Food Chemistry | Year: 2011

For implementation of the issued regulations and labeling policies for genetically modified organism (GMO) supervision, the polymerase chain reaction (PCR) method has been widely used due to its high specificity and sensitivity. In particular, use of the event-specific PCR method based on the flanking sequence of transgenes has become the primary trend. In this study, both qualitative and quantitative PCR methods were established on the basis of the 5′ flanking sequence of transgenic soybean A2704-12 and the 3′ flanking sequence of transgenic soybean A5547-127, respectively. In qualitative PCR assays, the limits of detection (LODs) were 10 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127. In quantitative real-time PCR assays, the LODs were 5 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127, and the limits of quantification (LOQs) were 10 copies for both. Low bias and acceptable SD and RSD values were also achieved in quantification of four blind samples using the developed real-time PCR assays. In addition, the developed PCR assays for the two transgenic soybean events were used for routine analysis of soybean samples imported to Shanghai in a 6 month period from October 2010 to March 2011. A total of 27 lots of soybean from the United States and Argentina were analyzed: 8 lots from the Unites States were found to have the GM soybean A2704-12 event, and the GM contents were <1.5% in all eight analyzed lots. On the contrary, no GM soybean A5547-127 content was found in any of the eight lots. These results demonstrated that the established event-specific qualitative and quantitative PCR methods could be used effectively in routine identification and quantification of GM soybeans A2704-12 and A5547-127 and their derived products. © 2011 American Chemical Society.


Shen K.,Shanghai Ocean University | Shen K.,Shanghai JiaoTong University | Li X.,Shanghai JiaoTong University | Li X.,GMO Detection Laboratory | And 5 more authors.
Journal of AOAC International | Year: 2010

Despite rapid developments in the detection techniques for genetically modified organisms (GMOs), the event-specific PCR method with high specificity is still the most used technique. In this study, event-specific simplex and duplex qualitative and quantitative detection systems were developed targeting the 3′ insertion site of GM maize SYN-E3272-5 (3272) construct. A reference molecule p3272 was constructed to act as positive control and as calibrator for quantitative analysis. The LOD for simplex and duplex qualitative PCR assays was 10 copies of p3272 control DNA. LOD and the LOQ for simplex and duplex quantitative PCR assays were 10 and 25 copies of p3272 DNA, respectively. Furthermore, four practical GM maize samples were quantified using the established simplex and duplex quantitative PCR systems by in-house validation. Results from five operators showed that the bias ranged from 3.44 to 17.24% in the simplex system and from 0.42 to 16.06% in the duplex system, respectively. These results demonstrated that the established event-specific simplex and duplex qualitative and quantitative PCR systems combined with the reference molecule p3272 are suitable for the detection of GM maize 3272 and its derived products.


Guo J.,Shanghai JiaoTong University | Yang L.,Shanghai JiaoTong University | Chen L.,Shanghai Normal University | Morisset D.,Slovenian National Institute of Biology | And 3 more authors.
Analytical Chemistry | Year: 2011

We describe the development of a novel combined approach for high-throughput analysis of multiple DNA targets based on multiplex Microdroplet PCR Implemented Capillary gel electrophoresis (MPIC), a two-step PCR amplification strategy. In the first step, the multiple target DNAs are preamplified using bipartite primers attached with universal tail sequences on their 5'-ends. Then, the preamplified templates are compartmentalized individually in the microdroplet of the PCR system, and multiple targets can be amplified in parallel, employing primers targeting their universal sequences. Subsequently, the resulting multiple products are analyzed by capillary gel electrophoresis (CGE). Using genetically modified organism (GMO) analysis as a model, 24 DNA targets can be simultaneously detected with a relative limit of detection of 0.1% (w/w) and absolute limit of detection of 39 target DNA copies. The described system provides a promising alternative for high-throughput analysis of multiple DNA targets. © 2011 American Chemical Society.


PubMed | Jinlin Academy of Agricultural science, Shanghai JiaoTong University, GMO Detection Laboratory and Zhejiang Academy of Agricultural Sciences
Type: | Journal: Food chemistry | Year: 2015

For transferring the event-specific PCR methods of genetically modified papaya Huanong No.1 to other laboratories, we validated the previous developed PCR assays of Huanong No.1 according to the international standard organization (ISO) guidelines. A total of 11 laboratories participated and returned their test results in this trial. In qualitative PCR assay, the high specificity and limit of detection as low as 0.1% was confirmed. For the quantitative PCR assay, the limit of quantification was as low as 25 copies. The quantitative biases among ten blind samples were within the range between 0.21% and 10.04%. Furthermore, the measurement uncertainty of the quantitative PCR results was calculated within the range between 0.28% and 2.92% for these ten samples. All results demonstrated that the Huanong No.1 qualitative and quantitative PCR assays were creditable and applicable for identification and quantification of GM papaya Huanong No.1 in further routine lab analysis.


Li X.,GMO Detection Laboratory | Li J.,East China University of Science and Technology | Zhang S.,GMO Detection Laboratory | He Y.,GMO Detection Laboratory | Pan L.,GMO Detection Laboratory
Journal of Agricultural and Food Chemistry | Year: 2013

To avoid fraudulent substitutions in fish markets, the proper methods are needed to test the authenticity of the ingredients. As a preferable methodology, a quantitative real-time polymerase chain reaction (qPCR) method was used in this study to identify species from the Salmonidae family based on the salmon growth hormone gene. Fish samples of six genera from the Salmonidae family were tested to identify the specificity, sensitivity, and applicability of the established method. Results showed that the method was highly specific for salmonid detection. Ct values were obtained only from 31 Salmonidae fish species samples. The relative and absolute limits of detection were 0.01% and 25 pg of genomic DNA, respectively, which could meet with the requirements of routine detections. To test the applicability of the method, the content of salmonid ingredients in 16 commercial food products was quantified from standard curves constructed from DNA of two Salmonidae species. The results revealed that the salmonid ingredient was detected in 12 samples, indicating that 25% of the labels are inauthentic. These results demonstrate that the developed qPCR method is suitable for identification of Salmonidae ingredients. © 2013 American Chemical Society.


Li X.,GMO Detection Laboratory | Wang X.,East China University of Science and Technology | Yang J.,GMO Detection Laboratory | Liu Y.,GMO Detection Laboratory | And 2 more authors.
BMC Biotechnology | Year: 2014

Background: To date, over 150 genetically modified (GM) crops are widely cultivated. To comply with regulations developed for genetically modified organisms (GMOs), including labeling policies, many detection methods for GMO identification and quantification have been developed.Results: To detect the entrance and exit of unauthorized GM crop events in China, we developed a novel quadruplex real-time PCR method for simultaneous detection and quantification of GM cotton events GHB119 and T304-40 in cotton-derived products (based on the 5′-flanking sequence) and the insect-resistance gene Cry2Ae. The limit of detection was 10 copies for GHB119 and Cry2Ae and 25 copies for T304-40. The limit of quantification was 25 copies for GHB119 and Cry2Ae and 50 copies for T304-40. Moreover, low bias and acceptable standard deviation and relative standard deviation values were obtained in quantification analysis of six blind samples containing different GHB119 and T304-40 ingredients.Conclusions: The developed quadruplex quantitative method could be used for quantitative detection of two GM cotton events (GHB119 and T304-40) and Cry2Ae gene ingredient in cotton derived products. © 2014 Li et al.; licensee BioMed Central Ltd.


Li X.,Bor Luh Food Safety Center | Li X.,GMO Detection Laboratory | Shen K.,Bor Luh Food Safety Center | Yang L.,Bor Luh Food Safety Center | And 3 more authors.
Food Control | Year: 2010

Plasmid molecule based reference material (RM) has been shown to be a good alternative as the calibrator for genetically modified organisms (GMOs) identification and quantification, while most of the currently developed plasmid RM can only be used for one specific target detection. In this study, a flexible plasmid RM pNK containing three DNA fragments, i.e. 5′ and 3′ event-specific sequences of maize NK603 and endogenous gene zSSIIb, was developed. We have proved that pNK is suitable for using as a calibrator in both 5′ and 3′ event-specific detection of maize NK603, compared with that of genuine genomic DNA. The limit of detection (LOD) was 10 copies of pNK DNA in conventional PCR assays. The absolute LOD and limit of quantification (LOQ) in quantitative PCR assays were 5 and 25 copies. The standard curves targeting to zSSIIb, 5′ and 3′ event-specific sequences based on pNK DNA showed high reaction efficiency and good linearity. Also, low bias and variations were obtained in practical samples quantification using pNK as the calibrator. These results demonstrated that the developed pNK is flexible and suitable for identification and quantification of maize NK603, as a preferable substitute of RM from the plant raw material. © 2009 Elsevier Ltd. All rights reserved.


PubMed | GMO Detection Laboratory
Type: Journal Article | Journal: Journal of agricultural and food chemistry | Year: 2012

For implementation of the issued regulations and labeling policies for genetically modified organism (GMO) supervision, the polymerase chain reaction (PCR) method has been widely used due to its high specificity and sensitivity. In particular, use of the event-specific PCR method based on the flanking sequence of transgenes has become the primary trend. In this study, both qualitative and quantitative PCR methods were established on the basis of the 5 flanking sequence of transgenic soybean A2704-12 and the 3 flanking sequence of transgenic soybean A5547-127, respectively. In qualitative PCR assays, the limits of detection (LODs) were 10 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127. In quantitative real-time PCR assays, the LODs were 5 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127, and the limits of quantification (LOQs) were 10 copies for both. Low bias and acceptable SD and RSD values were also achieved in quantification of four blind samples using the developed real-time PCR assays. In addition, the developed PCR assays for the two transgenic soybean events were used for routine analysis of soybean samples imported to Shanghai in a 6 month period from October 2010 to March 2011. A total of 27 lots of soybean from the United States and Argentina were analyzed: 8 lots from the Unites States were found to have the GM soybean A2704-12 event, and the GM contents were <1.5% in all eight analyzed lots. On the contrary, no GM soybean A5547-127 content was found in any of the eight lots. These results demonstrated that the established event-specific qualitative and quantitative PCR methods could be used effectively in routine identification and quantification of GM soybeans A2704-12 and A5547-127 and their derived products.


PubMed | GMO Detection Laboratory
Type: Evaluation Studies | Journal: Journal of agricultural and food chemistry | Year: 2013

To avoid fraudulent substitutions in fish markets, the proper methods are needed to test the authenticity of the ingredients. As a preferable methodology, a quantitative real-time polymerase chain reaction (qPCR) method was used in this study to identify species from the Salmonidae family based on the salmon growth hormone gene. Fish samples of six genera from the Salmonidae family were tested to identify the specificity, sensitivity, and applicability of the established method. Results showed that the method was highly specific for salmonid detection. Ct values were obtained only from 31 Salmonidae fish species samples. The relative and absolute limits of detection were 0.01% and 25 pg of genomic DNA, respectively, which could meet with the requirements of routine detections. To test the applicability of the method, the content of salmonid ingredients in 16 commercial food products was quantified from standard curves constructed from DNA of two Salmonidae species. The results revealed that the salmonid ingredient was detected in 12 samples, indicating that 25% of the labels are inauthentic. These results demonstrate that the developed qPCR method is suitable for identification of Salmonidae ingredients.

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