Development of event-specific qualitative and quantitative PCR detection methods for the transgenic maize BVLA430101
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The transgenic maize (Zea mays L.) line BVLA430101 overexpressing Aspergillus niger phyA2 was approved in 2009 to be planted in a given area in China. However, the flanking sequences and event-specific qualitative/quantitative PCR detection methods for this transgenic event have not been reported. In this study, we characterized the molecular features of the exogenous integration in BVLA430101 and developed event-specific qualitative/quantitative PCR detection methods. Using genome walking, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR), and conventional PCR assays, we revealed that one intact copy of phytase construct was integrated in the maize genome chromosome 3, which is followed by a fragment of the transformation vector containing partial sequence of pH2b promoter. The defined 3′ flanking sequence was 1644 bp in length, based on which the event-specific qualitative and quantitative PCR assays for BVLA430101 were developed. The limit of detection (LOD) of the qualitative PCR assay was 10 haploid genome copies, and the limits of detection and quantification (LOD and LOQ) of the quantitative PCR were 10 and 10 copies of maize haploid genome, respectively. In-house validation of the developed event-specific quantitative PCR method with three practical maize samples showed that the quantified biases between the test and true values ranged between 2.60 and 7.52 %. These results indicated that the developed event-specific qualitative and quantitative PCR methods based on the newly characterized 3′ flanking sequences can be used successfully for the identification and quantification of transgenic maize BVLA430101 and its derived products.
KeywordsIntegration flanking sequence Limit of detection Limit of quantification Phytase Qualitative and quantitative PCR Transgenic maize BVLA430101
We thank Dr. Rumei Chen from China Academy of Agricultural Sciences for supplying the seeds of transgenic maize BVLA430101 and its non-transgenic counterpart used in the present study.
This work was supported by the China National Transgenic Plant Special Fund (2013ZX08012-002 and 2014ZX08012-002), China Innovative Research Team, Ministry of Education, and the Programme of Introducing Talents of Discipline to Universities (111 Project, B14016), and Shanghai Leading Talent Project in Agriculture.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Compliance with ethics requirements
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee. This article does not contain any studies with animals performed by any of the authors.
Informed consent was obtained from all individual participants included in the study.
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