Potential of cross-priming amplification and DNA-based lateral-flow strip biosensor for rapid on-site GMO screening

The requirement to monitor the presence of genetically modified organisms (GMO) in a variety of marked products has generated an increasing demand for reliable, rapid, and time and cost-effective analytical methods. Here we report an on-site method for rapid detection of cauliflower mosaic virus promoter (CaMV 35S), a common element present in most GMO, using cross-priming amplification (CPA) technology. Detection was achieved using a DNA-based contamination-proof strip biosensor. The limit of detection was 30 copies for the pBI121 plasmid containing the CaMV 35S gene. The certified reference sample of GM maize line MON810 was detectable even at the low relative mass concentration of 0.05 %. The developed CPA method had high specificity for the CaMV 35S gene, as compared with other GM lines not containing this gene and non-GM products. The method was further validated using nine real-world samples, and the results were confirmed by real-time PCR analysis. Because of its simplicity, rapidity, and high sensitivity, this method of detecting the CaMV 35S gene has great commercial prospects for rapid GMO screening of high-consumption food and agriculture products.

[1]  A. Fire,et al.  Rolling replication of short DNA circles. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[2]  W. Powell,et al.  Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences , 2009, BMC biotechnology.

[3]  Hans-Ulrich Waiblinger,et al.  Collaborative trial validation studies of real-time PCR-based GMO screening methods for detection of the bar gene and the ctp2-cp4epsps construct. , 2009, Journal of agricultural and food chemistry.

[4]  Y. Devos,et al.  Detection of genetically modified plant products by protein strip testing: an evaluation of real-life samples , 2007 .

[5]  Dabing Zhang,et al.  Visual and Rapid Detection of Two Genetically Modified Soybean Events Using Loop-mediated Isothermal Amplification Method , 2010 .

[6]  R. Sooknanan,et al.  NASBA: A detection and amplification system uniquely suited for RNA , 1995 .

[7]  H. Akiyama,et al.  Evaluation of modified PCR quantitation of genetically modified maize and soybean using reference molecules: interlaboratory study. , 2009, Journal of AOAC International.

[8]  Eun Kyu Lee,et al.  Loop Mediated Isothermal Amplification of DNA , 2008 .

[9]  Yan Xu,et al.  Helicase‐dependent isothermal DNA amplification , 2004, EMBO reports.

[10]  T. Notomi,et al.  Development of a New Method for Diagnosis of Rubella Virus Infection by Reverse Transcription-Loop-Mediated Isothermal Amplification , 2006, Journal of Clinical Microbiology.

[11]  F. Liu,et al.  Rapid on-site detection of Acidovorax citrulli by cross-priming amplification. , 2012, Molecular and cellular probes.

[12]  M. Dickinson,et al.  Rapid detection of Phytophthora ramorum and P. kernoviae by two-minute DNA extraction followed by isothermal amplification and amplicon detection by generic lateral flow device. , 2010, Phytopathology.

[13]  P. Mariani,et al.  Potentials and limitations of molecular diagnostic methods in food safety , 2009, Genes & Nutrition.

[14]  C. Albrecht,et al.  Sensitive and highly specific quantitative real-time PCR and ELISA for recording a potential transfer of novel DNA and Cry1Ab protein from feed into bovine milk , 2009, Analytical and bioanalytical chemistry.

[15]  A. Fluit,et al.  False-Positive Results and Contamination in Nucleic Acid Amplification Assays: Suggestions for a Prevent and Destroy Strategy , 2004, European Journal of Clinical Microbiology and Infectious Diseases.

[16]  Z. Hongwei,et al.  Rapid and sensitive detection of Enterobacter sakazakii by cross-priming amplification combined with immuno-blotting analysis. , 2010, Molecular and cellular probes.

[17]  David Zilberman,et al.  Regulating agricultural biotechnology : economics and policy , 2006 .

[18]  Y. Mori,et al.  Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. , 2001, Biochemical and biophysical research communications.

[19]  Qian Gao,et al.  Cross-Priming Amplification for Rapid Detection of Mycobacterium tuberculosis in Sputum Specimens , 2009, Journal of Clinical Microbiology.

[20]  Nicolas Gryson,et al.  Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review , 2010, Analytical and bioanalytical chemistry.

[21]  T. Pan,et al.  Detection of genetically modified maize MON810 and NK603 by multiplex and real-time polymerase chain reaction methods. , 2004, Journal of agricultural and food chemistry.

[22]  Lin Hu,et al.  Cross Priming Amplification: Mechanism and Optimization for Isothermal DNA Amplification , 2012, Scientific Reports.

[23]  Rajesh Kumar,et al.  Development of nanocolloidal gold based immunochromatographic assay for rapid detection of transgenic vegetative insecticidal protein in genetically modified crops. , 2010 .

[24]  J. Mano,et al.  Real-time PCR array as a universal platform for the detection of genetically modified crops and its application in identifying unapproved genetically modified crops in Japan. , 2009, Journal of agricultural and food chemistry.

[25]  H. Kong,et al.  Application of isothermal helicase-dependent amplification with a disposable detection device in a simple sensitive stool test for toxigenic Clostridium difficile. , 2008, The Journal of molecular diagnostics : JMD.

[26]  R. Kumar.  Development of dipsticks for simultaneous detection of vip3A and cry1Ab/cry1Ac transgenic proteins. , 2012, Journal of AOAC International.

[27]  Li-li Hou,et al.  One-PCR-tube approach for in situ DNA isolation and detection. , 2011, The Analyst.

[28]  L L Wolfenbarger,et al.  The Ecological Risks of Engineered Crops , 1996 .

[29]  Farid E Ahmed,et al.  Detection of genetically modified organisms in foods. , 2002, Trends in biotechnology.

[30]  C. Carter,et al.  International Approval and Labeling Regulations of Genetically Modified Food in Major Trading Countries , 2006 .

[31]  T. K. Christopoulos,et al.  Dipstick test for DNA-based food authentication. Application to coffee authenticity assessment. , 2012, Journal of agricultural and food chemistry.

[32]  C. Yamada,et al.  A screening method for the detection of the 35S promoter and the nopaline synthase terminator in genetically modified organisms in a real-time multiplex polymerase chain reaction using high-resolution melting-curve analysis. , 2009, Biological & pharmaceutical bulletin.

[33]  Kozaburo Hayashi,et al.  Loop-Mediated Isothermal Amplification for Direct Detection of Mycobacterium tuberculosis Complex, M. avium, and M. intracellulare in Sputum Samples , 2003, Journal of Clinical Microbiology.

[34]  Hails,et al.  Genetically modified plants - the debate continues. , 2000, Trends in Ecology & Evolution.

[35]  G. Walker,et al.  Strand displacement amplification--an isothermal, in vitro DNA amplification technique. , 1992, Nucleic acids research.

[36]  J. Mano,et al.  Practicable group testing method to evaluate weight/weight GMO content in maize grains. , 2011, Journal of agricultural and food chemistry.

[37]  R. Kumar.  A quantitative immunopolymerase chain reaction method for detection of vegetative insecticidal protein in genetically modified crops. , 2011, Journal of Agricultural and Food Chemistry.