title: Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya (Carica papaya L.).
abstract: BACKGROUND
  Genetically engineered (GE) ringspot virus-resistant papaya cultivars 'Rainbow' and 'SunUp' have been grown in Hawai'i for over 10 years. In Hawai'i, the introduction of GE papayas into regions where non-GE cultivars are grown and where feral non-GE papayas exist have been accompanied with concerns associated with transgene flow. Of particular concern is the possibility of transgenic seeds being found in non-GE papaya fruits via cross-pollination. Development of high-throughput methods to reliably detect the adventitious presence of such transgenic material would benefit both the scientific and regulatory communities.
  
  
  RESULTS
  We assessed the accuracy of using conventional qualitative polymerase chain reaction (PCR) as well as real-time PCR-based assays to quantify the presence of transgenic DNA from bulk samples of non-GE papaya seeds. In this study, an optimized method of extracting high quality DNA from dry seeds of papaya was standardized. A reliable, sensitive real-time PCR method for detecting and quantifying viral coat protein (cp) transgenes in bulk seed samples utilizing the endogenous papain gene is presented. Quantification range was from 0.01 to 100 ng/μl of GE-papaya DNA template with a detection limit as low as 0.01% (10 pg). To test this system, we simulated transgene flow using known quantities of GE and non-GE DNA and determined that 0.038% (38 pg) GE papaya DNA could be detected using real-time PCR. We also validated this system by extracting DNA from known ratios of GE seeds to non-GE seeds of papaya followed by real-time PCR detection and observed a reliable detection limit of 0.4%.
  
  
  CONCLUSIONS
  This method for the quick and sensitive detection of transgenes in bulked papaya seed lots using conventional as well as real-time PCR-based methods will benefit numerous stakeholders. In particular, this method could be utilized to screen selected fruits from maternal non-GE papaya trees in Hawai'i for the presence of transgenic seed at typical regulatory threshold levels. Incorporation of subtle differences in primers and probes for variations in cp worldwide should allow this method to be utilized elsewhere when and if deregulation of transgenic papaya occurs.
authors.affiliation: Department of Plant Sciences, The University of Tennessee, 252 Ellington Plant Sciences, 2431 Joe Johnson Dr, Knoxville, TN 37996, USA. mnrao@utk.edu.
authors.forename: Madhugiri
authors.initials: M
authors.lastname: Nageswara-Rao
authors.forename: Charles
authors.initials: C
authors.lastname: Kwit
authors.forename: Sujata
authors.initials: S
authors.lastname: Agarwal
authors.forename: Mariah T
authors.initials: MT
authors.lastname: Patton
authors.forename: Jordan A
authors.initials: JA
authors.lastname: Skeen
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Yuan
authors.forename: Richard M
authors.initials: RM
authors.lastname: Manshardt
authors.forename: C Neal
authors.initials: CN
authors.lastname: Stewart
mesh_terms: D029441:Carica / Q000235:genetics*; D017931:DNA Primers; D018744:DNA, Plant / Q000235:genetics; D005638:Fruit / Q000235:genetics; D051456:Gene Flow; D005818:Genetic Engineering; D016014:Linear Models; D010935:Plant Diseases / Q000821:virology; D030821:Plants, Genetically Modified / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012639:Seeds / Q000235:genetics*; D012680:Sensitivity and Specificity; D019076:Transgenes
chemical_list: D017931:DNA Primers; D018744:DNA, Plant