Identification of GMO-DNA or animal components in food and feed has become an important application of public interest. In the novel food regulation, labelling has been mandatory for GMO containing food since they can be distinguished from conventional products by analytical methods, for example real-time PCR. Food samples are very heterogeneous and contain many different compounds like fat, cocoa or polysaccharides which can lead to suboptimal extraction or subsequent processing of DNA. NucleoSpin® Food guarantees good recovery rates even for small genomic DNA fragments (< 1 kb) out of processed, complex food matrices (e.g., ketchup or spices), which generally have very low DNA contents as well as poor quality, degraded DNA.
After the food samples have been homogenized, the DNA can be extracted with lysis buffers containing chaotropic salts, denaturing agents and detergents. The standard isolation ensures lysis using Buffer CF, which was especially developed in cooperation with GEN-IAL, Troisdorf, Germany (patent pending). Lysis mixtures should be cleared by centrifugation or filtration in order to remove contaminants and residual cellular debris. The clear supernatant is then mixed with binding buffer and ethanol to create conditions for optimal binding to the NucleoSpin® silica membrane, which was selected for this purpose due to its unique DNA-binding properties. After washing with two different buffers for efficient removal of potential PCR inhibitors, DNA can be eluted in low salt buffer or water, and is ready-to-use in subsequent reactions.
GMO, food and feed analysis with NucleoSpin® Food NucleoSpin® nucleic acid isolation technology from MACHEREY-NAGEL and GMO experience from GEN-IAL were combined to provide an optimal lysis and purification system for nearly all types of food samples. Resulting eluates are ready-to-use for all types of subsequent detection methods, especially real-time and basic PCR technology.
NucleoSpin® Food procedure
Application data
DNA analysis with LightCycler™ technology NucleoSpin® Food purified DNA from three matrices was amplified with lectin-specific primers and SYBR-Green detection mode in a LightCycler system (A). Specific amplification was verified by a melting point analysis (B).
Amplification of NucleoSpin® Food purified DNA DNA from soy meal was purified with NucleoSpin® Food and used for PCR amplification with lectin-specific (lanes1,2) and plant-specific (lanes 3,4) primer-pairs. M: marker
Soy lectin-specific PCR Indicated samples were purified with NucleoSpin® Food. Diluted aliquots of the eluates were amplified with lectin-specific primers resulting in a 200 bp fragment. Even from complex and processed matrices like chocolate amplifiable DNA could be purified
GMO quantification in cereals and chocolate Analysis of Roundup Ready Soya with LightCycler™ technology Genomic DNA was purified with NucleoSpin® Food from cereals (red) and chocolate (green). DNA content was quantified by amplification with soy lectin-specific primers (Fig. A). Blue curves represent a dilution series of soy standard DNA. Fig. B shows the second amplification of the purified DNA with GMO-specific primers and a dilution series of GMO-standard DNA for the quantification of GMO DNA content. Fig. C: calibration curve for quantification of Roundup Ready soy. Fig. D: corresponding melting curves of GMO-specific amplification.
Detection of bovine DNA in animal feed, spiked with bovine meal Cattle feed (29 % corn gluten, 7 % brewery residues, 25% citrus pulp, 13 % barley, 9 % soy meal and 15 % canola meal) was spiked with defined quantities of bovine meal, which had been heat-treated at 133 °C and 2.5 bar over 30 min. DNA was extracted with NucleoSpin® Food and amplified with the DNAnimal BOSIdent* kit from GeneScan for specific detection of bovine DNA. The system detects bovine meal at concentrations above 0.5 % (occasionally down to 0.1 %). The amplicon is derived from mitochondrial DNA and has a length of only 271 bp. Together with the highly efficient NucleoSpin® Food extraction system these characteristics allow the detection of bovine DNA at high sensitivity and specificity even in highly processed food and feed – like heat-treated animal meal.
*BOSIdent (catalog No. 5222103305) is a product of GeneScan Europe AG (www.genescan.com).
GMO analysis of spice samples Spice powder was processed with NucleoSpin® Food. The resulting eluates were analyzed for presence of DNA and GMO contents using LightCycler™ technology.
The results demonstrate that even from samples difficult to extract DNA can be purified with appropriate quality for subsequent real-time PCR analysis.
Beef detection in sausage products DNA preparation was done according to the NucleoSpin® Food standard protocol. Aliquots of the 100 μL eluates were amplified with primers and components of a commercial kit (CIBUS, Germany). Bovine DNA could be detected in several products, even in strongly processed samples.
Data kindly provided by GEN-IAL, Troisdorf, Germany
Sample 8 was declared to be prepared from duck meat only, but clearly showed presence of beef. Samples 3 and 7 did not contain detectable amounts of bovine DNA.
Identification of GMO-DNA or animal components in food and feed has become an important application of public interest. In the novel food regulation, labelling has been mandatory for GMO containing food since they can be distinguished from conventional products by analytical methods, for example real-time PCR. Food samples are very heterogeneous and contain many different compounds like fat, cocoa or polysaccharides which can lead to suboptimal extraction or subsequent processing of DNA. NucleoSpin® Food guarantees good recovery rates even for small genomic DNA fragments (< 1 kb) out of processed, complex food matrices (e.g., ketchup or spices), which generally have very low DNA contents as well as poor quality, degraded DNA.
After the food samples have been homogenized, the DNA can be extracted with lysis buffers containing chaotropic salts, denaturing agents and detergents. The standard isolation ensures lysis using Buffer CF, which was especially developed in cooperation with GEN-IAL, Troisdorf, Germany (patent pending). Lysis mixtures should be cleared by centrifugation or filtration in order to remove contaminants and residual cellular debris. The clear supernatant is then mixed with binding buffer and ethanol to create conditions for optimal binding to the NucleoSpin® silica membrane, which was selected for this purpose due to its unique DNA-binding properties. After washing with two different buffers for efficient removal of potential PCR inhibitors, DNA can be eluted in low salt buffer or water, and is ready-to-use in subsequent reactions.
GMO, food and feed analysis with NucleoSpin® Food NucleoSpin® nucleic acid isolation technology from MACHEREY-NAGEL and GMO experience from GEN-IAL were combined to provide an optimal lysis and purification system for nearly all types of food samples. Resulting eluates are ready-to-use for all types of subsequent detection methods, especially real-time and basic PCR technology.
NucleoSpin® Food procedure
Application data
DNA analysis with LightCycler™ technology NucleoSpin® Food purified DNA from three matrices was amplified with lectin-specific primers and SYBR-Green detection mode in a LightCycler system (A). Specific amplification was verified by a melting point analysis (B).
Amplification of NucleoSpin® Food purified DNA DNA from soy meal was purified with NucleoSpin® Food and used for PCR amplification with lectin-specific (lanes1,2) and plant-specific (lanes 3,4) primer-pairs. M: marker
Soy lectin-specific PCR Indicated samples were purified with NucleoSpin® Food. Diluted aliquots of the eluates were amplified with lectin-specific primers resulting in a 200 bp fragment. Even from complex and processed matrices like chocolate amplifiable DNA could be purified
GMO quantification in cereals and chocolate Analysis of Roundup Ready Soya with LightCycler™ technology Genomic DNA was purified with NucleoSpin® Food from cereals (red) and chocolate (green). DNA content was quantified by amplification with soy lectin-specific primers (Fig. A). Blue curves represent a dilution series of soy standard DNA. Fig. B shows the second amplification of the purified DNA with GMO-specific primers and a dilution series of GMO-standard DNA for the quantification of GMO DNA content. Fig. C: calibration curve for quantification of Roundup Ready soy. Fig. D: corresponding melting curves of GMO-specific amplification.
Detection of bovine DNA in animal feed, spiked with bovine meal Cattle feed (29 % corn gluten, 7 % brewery residues, 25% citrus pulp, 13 % barley, 9 % soy meal and 15 % canola meal) was spiked with defined quantities of bovine meal, which had been heat-treated at 133 °C and 2.5 bar over 30 min. DNA was extracted with NucleoSpin® Food and amplified with the DNAnimal BOSIdent* kit from GeneScan for specific detection of bovine DNA. The system detects bovine meal at concentrations above 0.5 % (occasionally down to 0.1 %). The amplicon is derived from mitochondrial DNA and has a length of only 271 bp. Together with the highly efficient NucleoSpin® Food extraction system these characteristics allow the detection of bovine DNA at high sensitivity and specificity even in highly processed food and feed – like heat-treated animal meal.
*BOSIdent (catalog No. 5222103305) is a product of GeneScan Europe AG (www.genescan.com).
GMO analysis of spice samples Spice powder was processed with NucleoSpin® Food. The resulting eluates were analyzed for presence of DNA and GMO contents using LightCycler™ technology.
The results demonstrate that even from samples difficult to extract DNA can be purified with appropriate quality for subsequent real-time PCR analysis.
Beef detection in sausage products DNA preparation was done according to the NucleoSpin® Food standard protocol. Aliquots of the 100 μL eluates were amplified with primers and components of a commercial kit (CIBUS, Germany). Bovine DNA could be detected in several products, even in strongly processed samples.
Data kindly provided by GEN-IAL, Troisdorf, Germany
Sample 8 was declared to be prepared from duck meat only, but clearly showed presence of beef. Samples 3 and 7 did not contain detectable amounts of bovine DNA.
