基于“遗传标志物”紫河车DNA指纹-试纸条分子鉴定方法及试剂的研究

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

目的:建立中药材紫河车遗传标志物DNA指纹-免疫胶体金试纸条快速分子鉴定方法,研发一体化质量控制基因检测试剂盒。方法:自主研发紫河车模板DNA提取的方法及试剂,根据人mtDNA cytb基因设计种属特异性引物,引物的5'端分别用FAM、Biotin标记,筛选PCR最佳退火温度及循环次数,建立DNA指纹分子鉴定方法,研发PCR基因检测试剂预混液,采用分子克隆及基因测序技术制备对照药材DNA克隆液,利用免疫胶体金试纸条进行结果检测,开发出集“DNA提取试剂、PCR基因检测试剂预混液、对照药材DNA克隆液、空白对照液、试纸条”为一体的快速基因检测试剂盒,并进行特异性、重现性、灵敏性、稳定性的评价。结果:自主研发的DNA提取试剂提取的模板DNA质量浓度均在150~280 ng·μL^(-1),纯度均在1.8~1.9,琼脂糖凝胶电泳无拖尾。紫河车特异性引物在退火温度59℃、循环20次时;免疫胶体金试纸条显示:紫河车对照药材及正品出现2条红色条带,易混品及空白对照出现1条红的条带。琼脂糖凝胶电泳验证正确。对照药材DNA测序结果与人mtDNA cytb基因同源性100%。自主研发的一体化快速基因检测试剂盒的特异性强,重现性好,稳定性好,灵敏度为0.1 ng·μL^(-1)。结论:DNA指纹-免疫胶体金试纸条分子鉴定方法能够特异、准确、快速、可视化地对紫河车的真伪进行鉴定,一体化快速基因检测试剂盒为紫河车质量控制提供规范化、标准化的检测方式,更适合现场实地检测,可普遍推广使用。

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.