Study on molecular identification methods and reagents “genetic marker”-based DNA fingerprinting-test strips of human placentophagy<sup>*</sup>

WANG Yan-shuang, WANG Yi-tong, MU Run-hong, ZHANG Li-hua

doi:10.16155/j.0254-1793.2023-0435

Chinese Journal of Pharmaceutical Analysis >

2024 , Vol. 44 >Issue 7: 1276 - 1284

DOI: https://doi.org/10.16155/j.0254-1793.2023-0435

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Study on molecular identification methods and reagents “genetic marker”-based DNA fingerprinting-test strips of human placentophagy^*

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1. Basic Medicine School, Beihua University, Jilin 132013, China;
2. Second Hospital,Jilin University, Changchun 130000, China;
3. Chinese Medicine DNA Fingerprint Detection Technology Technology Innovation Center, Beihua University, Jilin 132013, China;
4. Jilin Leining Food and Drug Testing Technology Service Co., Ltd., Jilin 132013, China

Received date: 2024-05-30

Online published: 2024-08-05

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Abstract

Objective: To establish a rapid molecular identification method for DNA fingerprinting-immunocolloidal gold test strips of Chinese herbal medicine human placentophagy genetic markers and to develop an integrated quality control gene detection kit. Methods: Self-developed method and reagents could be for template DNA extraction from human placentophagy. Species-specific primers were designed according to the human mtDNA cytb gene, and the 5' ends of the primers were labeled with FAM and Biotin, respectively. The optimal annealing temperature and cycle number of PCR were screened to establish a DNA fingerprinting molecular identification method. To develop PCR gene detection reagent premixes, to prepare DNA clones of control herbs using molecular cloning and gene sequencing techniques and to utilize immunocolloidal gold test strips for result detection. Finally, a rapid gene detection kit integrating DNA extraction reagent, PCR gene detection reagent premixes, control herb DNA clone solution, blank control solution and test strips was developed, and evaluated for specificity, reproducibility, sensitivity and stability. Results: The template DNA extracted by self-developed DNA extraction reagents were all in the concentration of 150-280 ng·μL^-1, the purities were all in the range of 1.8-1.9, and there were no trail in the agarose gel electrophoresis. And the immunocolloidal gold test strips showed two red bands for human placentophagy control herb and authentic product, and one red band for easy-mix product and blank control at annealing temperature of 59 ℃ and 20 cycles for human placentophagy specific primers. The agarose gel electrophoresis was verified to be correct. The DNA sequencing results of the control herbs were 100% homologous with human mtDNA cytb gene. The self-developed integrated rapid gene detection kit was specific, reproducible, stable and sensitive at 0.1 ng·μL^-1. Conclusion: The DNA fingerprinting-immunocolloidal gold test strip molecular identification method can specifically, accurately, rapidly and visually identify the authenticity of human placentophagy, and the integrated rapid gene detection kit provides a regulated and standardized testing method for quality control of human placentophagy. Therefore, it is more suitable for on-site field testing and universal promotion.

Key words： Placenta Hominis; genetic marker; mitochondrial cytochrome b; PCR technique; DNA fingerprinting; immunocolloidal gold test strip; integrated reagent kit

Cite this article

WANG Yan-shuang, WANG Yi-tong, MU Run-hong, ZHANG Li-hua . Study on molecular identification methods and reagents “genetic marker”-based DNA fingerprinting-test strips of human placentophagy^*[J]. Chinese Journal of Pharmaceutical Analysis, 2024 , 44(7) : 1276 -1284 . DOI: 10.16155/j.0254-1793.2023-0435

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