Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya (Carica papayaL.)
© Nageswara-Rao et al.; licensee BioMed Central Ltd. 2013
Received: 4 July 2013
Accepted: 27 August 2013
Published: 1 September 2013
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.
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%.
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.
KeywordsCoat protein (CP) Genetically-engineered Papain Quantitative polymerase chain reaction (qPCR) Seeds Transgene Virus resistance
Papaya (Carica papaya L.) is widely grown in tropical and subtropical regions for its nutritional benefits and medicinal applications. World production of papaya is approximately 11.5 million tons with the USA accounting for 13,653 tons . It is among the top 10 commodities produced in Hawai’i, USA with a farm gate value of $11.1 million in 2010 . It is a polygamous diploid (2n = 18) plant species with a complex breeding system including dioecious and gynodioecious forms that are manifested through individuals being male, female, or hermaphrodites [3, 4]. In Hawai’i, only the hermaphrodite plants are currently commercially important. Male and female papayas are obligate outcrossers, whereas hermaphrodites are self-pollinating. However, cross-pollination has been reported in hermaphrodite papayas at various levels depending upon a number of factors such as morphological relationships of stamens and stigma, timing of anther dehiscence relative to flower anthesis, and incidence of insect pollinators [5–8].
A major obstacle to large-scale commercial production of papaya worldwide is the devastating disease caused by papaya ringspot virus (PRSV), which severely impacts papaya yield [9–11]. Development of genetically engineered (GE) virus-resistant papaya was initiated in 1987 and culminated in 1998 with the commercial release of two GE cultivars, ‘Rainbow’ and ‘SunUp', which were transformed with the modified binary vector pGA482GG/cpPRV-4 carrying gus, nptII, and PRSV coat protein (cp) transgenes [12–15]. These have been widely planted in Hawai’i, with ‘Rainbow’ accounting for 77% of the total 805 ha in commercial production . The incorporation of GE papaya into the agricultural landscape in Hawai’i confers the possibility of movement of transgenes between the GE and non-GE papayas through outcrossing (i.e., pollen movement) or seed movement. Hence, there is a need for better information about the rates of gene flow in papaya to monitor and minimize adventitious presence of transgenes and facilitate profitable coexistence of GE and non-GE papaya growers. We are especially interested in gene flow via pollen wherein it is conceivable that transgenic seed could reside within fruit produced on a non-transgenic plant. This has motivated our effort to develop reliable methods for detection of transgene in a mixture of putatively GE and non-GE papaya seeds.
A number of different assays have been employed to detect transgene flow. Previously, the GUS marker gene was employed to track pollen movement from a 0.5 ha ‘Rainbow’ papaya field into surrounding border rows of non-GE papaya plants using histochemical GUS staining . Alternatively, real-time PCR (or qPCR) assays have been developed for several GE plant species for efficient transgene detection in mixed samples [17–20]. Real-time PCR offers a quick, economical and high-throughput alternative for detection of gene flow in GE and non-GE plants as compared to the GUS assay, Southern blot or conventional PCR analysis. Real-time PCR of bulk DNA extractions from seed has been utilized to detect adventitious presence of transgenes in maize . This technique is probably the most appropriate for detecting transgenes in bulked seed lots of papaya. The development of a real-time PCR detection method for assessing transgenic status of papaya using papain and cp genes has been reported . However, the researchers did not report the use of this method on a mixture of GE and non-GE papaya. Thus, our goal was to improve the published method  for detection in mixed (GE and non-GE) samples as well as optimize DNA extraction from dried papaya seeds. We envisage our methodology as being helpful to detect adventitious presence of transgenes in mainly non-GE papaya seed lots. In the present study, we extracted DNA from bulked dry seeds and then utilized conventional and real-time PCR assays to test transgene detection limit in GE papaya, as well as known mixtures of DNAs from GE and non-GE papaya. We also investigated transgene detection limit in GE and non-GE papaya in different ratios of GE and non-GE papaya seed mixtures. Since papaya seeds are rich in polysaccharides and preliminary experiments showed that reliable DNA extraction required optimization for maximal sensitivity of detection, we performed detailed experiments using various DNA isolation procedures. The goal of this research was to produce a protocol that could be reliably used to estimate GE seed presence within papaya fruits, with special attention to predominantly non-GE bulk samples.
We used non-GE ‘Waimanalo’ papaya seeds and GE seeds containing PRSV cp transgene from the cultivars ‘SunUp’ (homozygous CP/CP) and ‘Rainbow’ (hemizygous CP/-). All seed samples were used for genomic DNA extraction, as well as conventional PCR and real-time PCR procedures.
DNA extraction and quantification optimization
Genomic DNA from 500 mg dry seeds (~45 seeds) was extracted by the following six methods to determine which one was optimal and most reliable for PCR: (1) DNeasy Plant Mini kit (Qiagen Inc., Valencia, CA, USA), (2) TRIzol reagent method (Life Technologies, Carlsbad, CA, USA), (3) QIAcube kit (Qiagen Inc., Valencia, CA, USA), (4) Promega Maxwell 16 kit (Promega, Madison, WI, USA), (5) CTAB method , and (6) modified CTAB method. For each method, three independent experiments were performed incorporating three types of papaya samples (‘Waimanalo', ‘Rainbow’ and ‘SunUp’). Seeds were macerated using a mortar and pestle under liquid nitrogen. Protocols for the commercial DNA isolation kits were followed according to the manufacturers’ procedures. In addition, the extracted genomic DNA was treated with 4 μl of RNaseA (10 mg/ml; Fisher Scientific, Pittsburgh, PA, USA).
We sought to optimize a CTAB method  (modified CTAB) using the following extraction procedure. Seeds were macerated using a mortar and pestle under liquid nitrogen wherein 5 ml extraction buffer (100 mM Tris–HCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 2% CTAB, 1% PVP-40, 1% PVPP-40 and 2% β-mercaptoethanol) was added. The samples were incubated at 65°C for 45 min (with intermittent inversion every 10 min). To improve the quality of extracted genomic DNA, the suspension was emulsified with equal volume of phenol (pH 5.0): chloroform: isoamyl alcohol (25:24:1; biotechnology grade, Fisher Scientific, Pittsburgh, PA, USA) twice. This was followed by emulsification with equal volume of chloroform: isoamyl alcohol (24:1) step performed twice. Genomic DNA was precipitated by addition of two volumes of chilled isopropanol, and the pellet was washed twice with 250 μl of chilled 70% ethanol prior to suspension in 100 μl of TE buffer [10 mM Tris–HCl (pH 8.0), 1 mM EDTA (pH 8.0)]. The extracted genomic DNA was treated with 4 μl of RNaseA (10 mg/ml; Fisher Scientific, Pittsburgh, PA, USA) to completely remove the residual RNA. Genomic DNA was again re-precipitated with two volumes of chilled isopropanol and 1/10th volume of 7.5 M sodium acetate to remove residual polysaccharides from DNA, and the pellet was washed with 100 μl of chilled 70% ethanol before re-suspension in 50 μl of TE buffer [10 mM Tris–HCl (pH 8.0), 1 mM EDTA (pH 8.0)] to increase the yield. Genomic DNA concentration was determined by using a Nanodrop ND1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) as well as by electrophoresis in 1% agarose gels with 1× TAE buffer (pH 8.0) with detection under UV light after ethidium bromide staining. DNA was also extracted from GE and non-GE papaya seed mixtures at 10:90 (10%), 1:99 (1%), 1:249 (0.4%), 1:499 (0.2%) and 1:999 (0.1%).
Primer pairs and fluorogenic probes used for the conventional and real time-PCR
Primers and probes
Sequence (5' →3')
Amplification length (bp)
GGC TCA ATA TGG TAT TCA CTA CAG AAA T
CAT CGG TTT TGG CTG CAT AA
GAC ATC TCT AAC ACT CGC GC
CTT CGA GAG CCA TAT CAG GTG
AGT GGC TCA ATA TGG TAT TCA CTA CAG A
AAA ATG TAG ATA TAC CTC CCT TGA GCG
(FAM)-ATA CTT ACC CAT ATG AGG GAG TGC AAC GTT ATT G-(TAMRA)
CCG CGG TAT GGA ATC AAG AG
TCG AGA GCC ATA TCA GGT GTT TT
(FAM)-CTC GCT AGA TAT GCT TTC GAT TTC TAT GCG GT-(MGB)
Gene-specific (Table 1) non-fluorescent forward and reverse primer pairs for papain and cp genes (papain-B1 and papain-B2, and CP-B1 and CP-B2 respectively ), along with TaqMan® fluorescent dye-labeled probes for papain and cp genes were synthesized by Applied Biosystems (Foster City, USA). Papain and CP probes were both labeled with FAM (6-fluorescein amidite) fluorescent reporter dye at the 5' end. At the 3’ end, the papain probe was labeled with fluorescent quencher dye 6-carboxytetramethylrhodamine (TAMRA), while the CP probe was labeled with minor groove binding (MGB) dye. The papain gene was used as an internal control  to optimize the quality of DNA extracted by various methods and for assessing the efficiency of real-time PCR for the selected cp transgene.
Real-time PCR, performed in a 96-well optical reaction plate (Applied Biosystems, Foster City, USA), containing a 20 μl reaction mixture of 1× TaqMan universal PCR master mix (includes ROX as a passive reference dye), 0.9 μM each of forward and reverse primers, 0.4 μM probe and 2.5 μl of respective DNA solution. For the generation of a standard curve, the extracted DNA was serially diluted to final concentrations of 100, 10, 1.0 and 0.01 ng/μl. Real-time PCR (ABI7900 Fast Real-time PCR system; Applied Biosystems, Foster City, USA) was performed using the following program: 50°C for 2 min, 95°C for 10 min, 45 cycles of 95°C for 15 s, 58°C 30 s, and 60°C for 30 s. Real-time PCR products were also resolved on 2% agarose gels with 1× TAE buffer (pH 8.0) and were detected under UV light after ethidium bromide staining. A standard regression curve of Ct values generated from DNA samples of known concentrations was interpolated for quantification. All reactions were performed in triplicate with papain primers and water as internal controls.
Validation: sensitivity of real-time PCR assays in a range of dilutions of GE and non-GE papaya seed DNA
A dilution series involving mixtures of GE and non-GE papaya genomic DNA was used to validate the sensitivity of real-time PCR assays in detecting the presence of transgenes. We mixed GE papaya genomic DNA with non-GE papaya genomic DNA such that the % GE DNA material constituted 50, 25, 12.5, 6.25, 3.125, 1.56, 0.75 and 0.038% of total DNA. Mixtures of GE and non-GE papaya seeds at 10, 1, 0.4, 0.2 and 0.1% were also utilized for real-time PCR assay. Standard regression curves of Ct (cycle threshold) values generated from DNA samples of known concentrations and seed mixtures were interpolated to estimate transgene quantities. All reactions were performed in triplicate with papain primers and water as internal controls.
Results and discussion
Concerns over the use of GE organisms have led to myriad national regulations for transgenic plants in most countries. Labeling of GE food products has become an important part of the regulatory framework in many countries, including those in the European Union, United Kingdom, Japan, Australia, New Zealand, and Thailand [24, 25]. In Hawai’i, because of close proximity of commercial fields of conventional and GE papaya plants, a situation exists in which adventitious presence of transgenes might occur at low frequency in non-GE fields [16, 26, 27]. Consequently, until recently, shipment of non-GE papayas from Hawai’i to Japan required a cumbersome “Identity Preservation Protocol” involving certification of non-GE status of each papaya tree using GUS assays [28, 29]. Of course, a GE pollination event onto a non-GE tree could yield GE seed. The USDA Tropical Plant Genetic Resources and Disease Research (TPGRDR) unit in Hilo, Hawai’i, is also concerned about the accidental export of adventitious transgenes in papaya germplasm provided to overseas research or industry destinations . Hence, there is a clear need for higher throughput and reliable methods for detection, identification and tracking of transgenes. Of particular utility would be procedures that could use DNA extracted from bulked tissue samples, especially seeds. Such methods will be of real benefit to the state and national government agencies charged with regulating shipments of GE products for commercial or research purposes.
The comparison of genomic DNA purity and yield in dry papaya seeds using various DNA extraction methods
Purity A260/280 A260/230
DNeasy Plant mini kit
Promega Maxwell 16
Absorbance of each papaya genomic DNA sample was evaluated at the ratios A260/A280 and A260/A230 and the purity and yield of genomic DNA are presented in Table 2. It is generally regarded that ratio A260/A280 values of 1.8 indicate high purity DNA, whereas less than 1.8 indicate protein contamination in DNA samples, and more than 1.8 indicate that there might be RNA contamination . The resultant A260/A280 and A260/A230 absorbance ratios were 1.82 and 1.76 respectively in the modified CTAB method, indicating that the papaya seed genomic DNA was free of protein and polysaccharides/polyphenol contamination (Table 2) and the quantity and quality of genomic DNA was suitable for both conventional as well as real-time PCR amplifications.
The present study adds to PCR  and real-time  PCR detection methods that have been developed to detect transgenes in papaya using papain and cp genes. Xu et al.  tested papaya varieties to confirm the presence of papain gene for use as an appropriate internal control gene. They established levels of detection (10 pg) of DNA as template for papain and cp gene to confirm the transgenic nature of GE papaya varieties utilized in their study , which was validated in the present study using same primer sequences. Similar detection levels with linear relationships and slope values were also obtained in the current study, however we used different germplasm . Unlike previous studies, these results were validated by using known amounts of GE and non-GE papaya in DNA mixtures whereby the transgenic DNA could be detected as low as 0.038%. Furthermore, the present study was also validated by assaying known amounts of transgenic seed in mixtures to determine sensitivity down to 0.4% transgenic seed at the hemizygous state. In both these experiments, detection levels (0.038% and 0.4%) were below threshold typical of regulatory agencies.
Absence of quantitative data on the incidence of adventitious transgene presence has led to speculation on the consequences of biological risks in papaya. We have developed a procedure to quantify and describe adventitious presence of GE transgenes in mixtures of GE and non-GE papaya seeds. This real-time PCR based detection technique should be useful for quick and sensitive detection of GE vs non-GE papaya as a biosafety and regulatory tool.
We wish to dedicate this paper to deceased papaya biologist Dr. T.N. Prabha, former mentor of the first author. This project was supported by Biotechnology Risk Assessment Grant Program from the USDA National Institute of Food and Agriculture. We appreciate funding assistance from the University of Tennessee Institute of Agriculture as well.
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