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Selection of a Taxon-Specific Reference Gene for Qualitative and Quantitative PCR Detection of Carthamus tinctorius

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Abstract

Safflower (Carthamus tinctorius) is an emerging model plant for the transgenic modification of fatty acid composition and the production of pharmaceuticals, proteins, or enzymes. Safflower is also a traditional Chinese medicine and is often used as a fake saffron product. Reliable detection of an endogenous reference gene is indispensable for the supervision of genetically modified safflower. Such an endogenous reference gene can also be used to specifically identify safflower ingredient in complex mixtures such as medicine or food. In this study, we identified and validated the CTOS gene as an endogenous reference for safflower. Conventional and real-time polymerase chain reaction (PCR) methods for detecting the CTOS gene sequence showed high interspecies specificity and intra-species stability. The lowest copy number detectable by conventional PCR was 10 haploid copies. The limit of detection and limit of quantification for the real-time PCR assay were estimated to be five and 40 haploid genome copies, respectively. Standard curves established for the real-time PCR assay exhibited good linearity (R 2 > 0.99) between the cycle threshold (Ct) values and the initial template copies. The developed conventional and real-time PCR assays were validated in routine analysis of the safflower ingredient in commercial Chinese medicines. In conclusion, the developed quantitative PCR methods were sufficiently specific and sensitive to be used in safflower genomic DNA quantification and safflower ingredient identification.

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Acknowledgements

We would like to thank Dr. Waltraud Schulze and Dr. Ossi Turunen for their help in preparing the manuscript. This research was supported by grants from a project of the National Natural Science Foundation of China (31401607) and a project of Natural Science Foundation of Hubei Province of China (2016CFB616).

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Authors and Affiliations

  1. Hubei Provincial Key Laboratory for the Protection and Application of Special Plants in Wuling Area of China, College of Life Science, South-Central University for Nationalities, 182 Min-Yuan Road, Wuhan, 430074, China

    Li Zhang, He Zhu, Jie Ke & Rui Qin

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  1. Li Zhang
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  2. He Zhu
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Correspondence to Rui Qin.

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Li Zhang declares that she has no competing interests. He Zhu declares that he has no competing interests. Jie Ke declares that he has no competing interests. Rui Qin declares that he has no competing interests.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Li Zhang and He Zhu contributed equally to this work

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Zhang, L., Zhu, H., Ke, J. et al. Selection of a Taxon-Specific Reference Gene for Qualitative and Quantitative PCR Detection of Carthamus tinctorius . Food Anal. Methods 10, 2952–2963 (2017). https://doi.org/10.1007/s12161-017-0855-9

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