Establishment of Real-time Polymerase Chain Reaction-based Molecular Markers to Distinguish between Rubus coreanus and Rubus occidentalis

Yo Ram Uh; Cheol Seong Jang

doi:10.22698/jales.20230002

All Issue

2023 Vol.35, Issue 1 Preview Page Next Page

Research Article

Establishment of Real-time Polymerase Chain Reaction-based Molecular Markers to Distinguish between Rubus coreanus and Rubus occidentalis

31 March 2023. pp. 14-25

PDF XML

Abstract

Rubus coreanus and Rubus occidentalis of the Rosaceae family are deciduous shrubs, which are commonly used in various health functional foods. The scientific name and origin of R. coreanus and R. occidentalis are distinct. However, both plants are often confused due to the morphological similarity of their fruits and misuse of the common name of R. occidentalis, “Bokbunja,” in Korea. In the present study, molecular markers were developed using quantitative real-time polymerase chain reaction (PCR) to differentiate between the two species. Species-specific primers were designed using the internal transcribed spacer region of ribosomal DNA and chloroplast genes, including matK, atpA, rpoB, and rpoC2. When the six designed primer sets were verified, the slope ranged from -3.18 to -3.55 and the correlation coefficient exceeded 0.99. Accordingly, the efficiency ranged between 91.10% and 106.27%. Furthermore, the developed markers were verified using a specificity test on 14 other species, including the target species. The developed real-time PCR-based molecular markers are expected to prevent the misuse of R. coreanus and R. occidentalis, ultimately contributing to the protection of consumer rights in the medical herb market.

Keywords

DNA markers

Real-time PCR

Rubus coreanus

Rubus occidentalis

Species identification

References

Arya, M., Shergill, I. S., Williamson, M., Gommersall, L., Arya, N., Patel, H. R. (2005) Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn 5:209-219. 10.1586/14737159.5.2.20915833050

Ekor, M. (2013) The growing use of herbal medicines: Issues relating to adverse reactions and challenges in monitoring safety. Front Neurol 4:177. 10.3389/fphar.2013.0017724454289PMC3887317

European Network of GMO Laboratories (ENGL) (2008) Definition of minimum performance requirements for analytical methods of GMO testing.

Hong, E., Lee, S. Y., Jeong, J. Y., Park, J. M., Kim, B. H., Kwon, K., Chun, H. S. (2017) Modern analytical methods for the detection of food fraud and adulteration by food category. J Sci Food Agric 97:3877-3896. 10.1002/jsfa.836428397254

Kane, D. E., Hellberg, R. S. (2016) Identification of species in ground meat products sold on the US commercial market using DNA-based methods. Food Control 59:158-163. 10.1016/j.foodcont.2015.05.020

Kim, L. S., Youn, S. H., Kim, J. Y. (2014) Comparative study on antioxidant effects of extracts from Rubus coreanus and Rubus occidentalis. J Korean Soc Food Sci Nutr 43:1357-1362. 10.3746/jkfn.2014.43.9.1357

Kim, Y. D., Uh, Y. R., Jang, C. S. (2022) Development of real-time PCR based molecular markers for two medicinal herb Artemisia species A. capillaris and A. iwayomogi. Food Sci Biotechnol 1-11. 10.1007/s10068-022-01166-036606092

Lo, Y. T., Shaw, P. C. (2018) DNA-based techniques for authentication of processed food and food supplements. Food Chemistry 240:767-774. 10.1016/j.foodchem.2017.08.02228946341

Minas, K., McEwan, N. R., Newbold, C. J., Scott, K. P. (2011) Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures. FEMS Microbiology Letters 325:162-169. 10.1111/j.1574-6968.2011.02424.x22029887

Ministry of Food and Drug Safety (2022) The dispensatory on the visual and organoleptic examination of herbal medicine. 71-72, 80-81.

Oh, S. H., Jang, C. S. (2020). Development and validation of a real-time PCR based assay to detect adulteration with corn in commercial turmeric powder products. Foods 9:882. 10.3390/foods907088232635672PMC7404567

Oh, S. H., Kim, Y. D., Jang, C. S. (2022) Development and application of DNA markers to detect adulteration with Scopolia japonica in the medicinal herb Atractylodes lancea. Food Sci Biotechnol 31:89-100. 10.1007/s10068-021-01008-535059233PMC8733082

Patel, A. V., Rojas-Vera, J., Dacke, C. G. (2004) Therapeutic constituents and actions of Rubus species. Curr Med Chem 11:1501-1512. 10.2174/092986704336514315180580

Safdar, M., Junejo, Y. (2015) Development and validation of fast duplex real-time PCR assays based on SYBER Green florescence for detection of bovine and poultry origins in feedstuffs. Food Chemistry 173:660-664. 10.1016/j.foodchem.2014.10.08825466073

Şakalar, E., Kaynak, A. (2016) Practical Molecular Detection Method of Beef and Pork in Meat and Meat Products by Intercalating Dye Based Duplex Real-Time Polimerase Chain Reaction. Int J Food Prop 19:31-40. 10.1080/10942912.2015.1017049

Seo, J. M., Ahn, J. Y. (2004) Biological activities of rosaceae plants extracts. J Soc Cosmet Scientists Korea 30:499-502.

Uh, Y. R., Jang, C. S. (2022) Establishing DNA markers to differentiate Agastache rugosa and Pogostemon cablin, which are confusedly used as medicinal herbs, using real-time PCR. Food Sci Biotechnol 1-9. 10.1007/s10068-022-01176-y36647523

World Health Organization (WHO) (2002) Traditional Medicine Strategy 2002-2005: Geneva, Switzerland. WHO/EDM/TRM/2002.1.

World Health Organization (WHO) (2007) Health of Indigenous Peoples. Geneva, p.1132.

Xu, W., Zhai, Z., Huang, K., Zhang, N., Yuan, Y., Shang, Y., Luo, Y. (2012) A novel universal primer-multiplex-PCR method with sequencing gel electrophoresis analysis. PloS one 7:e22900. 10.1371/journal.pone.002290022272223PMC3260127

Yang, H. M., Lim, S. S., Lee, Y. S., Shin, H. K., Oh, Y. S., Kim, J. K. (2007) Comparison of the anti-inflammatory effects of the extracts from Rubus coreanus and Rubus occidentalis. Korean J Food Sci Technol 39:342-347.

Yuan, J. S., Reed, A., Chen, F., Stewart, C. N. (2006) Statistical analysis of real-time PCR data. BMC bioinformatics 7:1-12. 10.1186/1471-2105-7-8516504059PMC1395339

Information

Publisher :Agriculture and Life Sciences Research Institute, Kangwon National University
Publisher(Ko) :강원대학교 농업생명과학대학 농업생명과학연구원
Journal Title :Journal of Agricultural, Life and Environmental Sciences
Journal Title(Ko) :농업생명환경연구
Volume : 35
No :1
Pages :14-25
Received Date : 2023-01-13
Revised Date : 2023-02-21
Accepted Date : 2023-02-22
DOI :https://doi.org/10.22698/jales.20230002

Journal of Agricultural, Life and Environmental SciencesISSN:2233-8322(Print) 2508-870X(Online)강원대학교 농업생명과학대학 농업생명과학연구원

All Issue