Event-specific PCR Research Articles

Development and Evaluation of Event-Specific Qualitative PCR Methods for Genetically Modified Bt10 Maize

In 2005 it was reported that the genetically modified (GM) maize strain or "event" called Bt10 had been distributed inadvertently in the United States over the previous 4 years. In order to ensure that grain for food and feed production did not contain trace amounts of Bt10 maize and complied with the applicable regulation, highly sensitive and specific detection of Bt10 maize was required. Accordingly, we developed a novel qualitative PCR system for specific detection of Bt10 maize. Moreover, we amply evaluated the performance characteristics of two PCR systems, our own and the one provided by the developer of Bt10, Syngenta Co. Ltd. It was confirmed that both of the qualitative PCR systems can specifically detect Bt10 maize, and the results of a single-laboratory examination suggested that the limit of detection was approximately less than 0.05% for both methods. To evaluate the reproducibility of the methods, we organized an interlaboratory study with the participation of 6 laboratories and analysis of 240 blind test samples. In this paper, we report, for the first time, the statistical analysis of the qualitative PCR data obtained from the interlaboratory study. The results of this analysis also revealed that there was no significant difference in the sensitivity between the two aforementioned methods and that the limit of detection of both the methods was less than 0.05%. Thus, we conclude that both of the methods are equally suitable for correct identification and sensitive detection of the unapproved GM maize Bt10 event in test samples.

Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531

Based on the DNA sequences of the junctions between recombinant and cotton genomic DNA of the two genetically modified (GM) cotton varieties, herbicide-tolerance Mon1445 and insect-resistant Mon531, event-specific primers and probes for qualitative and quantitative PCR detection for both GM cotton varieties were designed, and corresponding detection methods were developed. In qualitative PCR detection, the simplex and multiplex PCR detection systems were established and employed to identify Mon1445 and Mon531 from other GM cottons and crops. The limits of detection (LODs) of the simplex PCR were 0.05% for both Mon1445 and Mon531 using 100 ng DNA templates in one reaction, and the LOD of multiplex PCR analysis was 0.1%. For further quantitative detection using TaqMan real-time PCR systems for Mon1445 and Mon531, one plasmid pMD-ECS, used as reference molecule was constructed, which contained the quantitative amplified fragments of Mon1445, Mon531, and cotton endogenous reference gene. The limits of quantification (LOQs) of Mon1445 and Mon531 event-specific PCR systems using plasmid pMD-ECS as reference molecule were 10 copies, and the quantification range was from 0.03 to 100% in 100 ng of the DNA template for one reaction. Thereafter, five mixed cotton samples containing 0, 0.5, 0.9, 3 and 5% Mon1445 or Mon531 were quantified using established real-time PCR systems to evaluate the accuracy and precision of the developed real-time PCR detection systems. The accuracy expressed as bias varied from 1.33 to 8.89% for tested Mon1445 cotton samples, and from 2.67 to 6.80% for Mon531. The precision expressed as relative standard deviations (RSD) were different from 1.13 to 30.00% for Mon1445 cotton, and from 1.27 to 24.68% for Mon531. The range of RSD was similar to other laboratory results (25%). Concluded from above results, we believed that the established event-specific qualitative and quantitative PCR systems for Mon1445 and Mon531 in this study are acceptable and suitable for GM cotton identification and quantification.

Characterisation of the 5? integration site and development of an event-specific real-time PCR assay for NK603 maize from a low starting copy number

Genetically modified (GM) maize event NK603 is in the pipeline for authorisation in the EU and is likely to become a dominating GM maize on the world market. The 5′ integration junction was characterised from a small quantity of commercially available genomic DNA. Specific primers and a probe were designed targeting the 5′ integration junction in the plant, amplifying a 102 bp DNA fragment. A method for specific detection and quantification of NK603 using event-specific real-time PCR based on the identified and cloned sequence is described. The assay was specificity tested against five different GM maizes, one non-GM maize and one GM soya bean. Plasmids were constructed for use as external standards in calibration curves. Due to the lack of certified or other suitable commercial reference material for quantification of NK603, the quantifications were performed on non-target DNA spiked with plasmid DNA containing the cloned NK603 5′ integration junction. The plasmid was detectable down to one copy per PCR. The limits of detection (LOD) and quantitation (LOQ) are estimated to be comparable to those of state-of-the-art methods applied for other GM events.

Qualitative and event-specific PCR real-time detection methods for StarLink maize

In this paper we present qualitative detection and identification methods for StarLink maize (event CBH-351). The methodology proposed envisages detection of an internal target site in the cry9c coding region, as well as two event-specific target sites at the junction between the CBH-351 insert DNA and the genomic plant DNA. The cry9c-specific primer pair, generating a 180 bp amplicon, has been tested and optimised for conventional end-point PCR amplification. The event-specific primer pairs, generating amplicons of 138 bp and 100 bp respectively, give good performance in a conventional end-point PCR and in a real-time PCR assay. Our results clearly demonstrate that the primer pairs proposed can be used in an unambiguous and specific PCR identification assay for StarLink maize.

Event specific real-time quantitative PCR for genetically modified Bt11 maize (Zea mays)

We report the characterisation of the 3'-integration junction between host plant DNA and integrated gene-construct of the genetically modified Bt11 maize, and the successive development and first validation of a transformation event-specific quantitative TaqMan 5'-nuclease PCR assay for detection and quantitation of Bt11 maize on the LightCycler. The transgenic DNA in Bt11 is inserted in a tandem repeated DNA sequence motif of approximately 180 bp, and we discuss some of the problems this creates for design, development and validation of event-specific PCR assays. The limit of detection of the PCR assay was estimated to 10 initial template copies. The limit of quantitation in pure maize materials was estimated to approximately 40 template copies. In food samples containing high levels of exogenous DNA, the impact of this DNA was estimated to increase the limit of quantitation to approximately 100 initial template copies. Because of the low number of template copies required for detection and quantification, in combination with small size of the amplicon (~100 bp), the assay may be applied to analyses of raw materials as well as processed food and feed.

Roundup Ready ® soybean event-specific real-time quantitative PCR assay and estimation of the practical detection and quantification limits in GMO analyses

An event-specific real-time PCR method for detection and quantification of genetically modified Roundup Ready soybean with TaqMan chemistry on the LightCycler, targeting the nopaline synthase terminator (3') junction between recombinant and host plant DNA is described. We distinguish between three types of detection and quantification limits: the absolute limits (referring to the initial number of template copies in the PCR), the relative limits (referring to the relative percentage of initial template copies of the recombinant sequence to copies of the haploid soybean genome that is detected), and the practical limits (referring to what is applicable in the PCR with the DNA that is being analysed). The absolute detection limit was determined to be a single initial template copy, while the absolute quantification limit was determined to be approximately 30 initial template copies. We discuss the relative and practical limits, and provide guidelines to estimating the practical limits.

Use of cloned DNA fragments for event-specific quantification of genetically modified organisms in pure and mixed food products

An event-specific PCR method for detection and quantification of genetically modified Roundup Ready soybean (RRS) is described in this article. The complete DNA sequence at both the right and left integration sites of this genetically modified organism has recently been determined. Based on these sequence data, transformation event-specific primer pairs were developed. These primers amplify a fragment of the unique junction region between the inserted DNA and the plant DNA and therefore act as unique identifiers. Two sensitive, qualitative PCR assays gave absolute detection limits of 5 copies of the RRS junction fragment in 100 pg of total DNA per reaction. A real-time PCR method was then developed with the LightCycler System. For determination of the RRS content, a completely new type of external calibration standard is introduced here. A fragment of the RRS specific junction region and a fragment of the endogenous soybean lectin gene were both cloned in a plasmid vector. These new diagnostic DNA fragments allow quantification of RRS in whichever type of matrix, in a range of 10–106 copies of each target.