中药材蛤蚧PCR-试纸条快速分子鉴定方法及检测试剂的研究

Rapid Molecular Identification Method and Detection Reagents of Chinese Herbal Medicine Gecko by PCR-Test Strips

目的建立中药材蛤蚧聚合酶链反应(PCR)-免疫胶体金试纸条快速分子鉴定方法,研发快速基因检测试剂盒。方法筛选提取基因组DNA的方法及试剂,根据蛤蚧mtDNA cytb基因设计种属特异性引物并标记,筛选最佳反应条件,研发PCR基因检测试剂,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。进而,开发出一体化快速基因检测试剂盒,并对特异性、重现性、灵敏性、稳定性进行评价。结果基因组DNA的完整性、浓度、纯度均满足PCR的要求,引物在退火温度63℃时,循环20次时,免疫胶体金试纸条显示:蛤蚧对照药材及正品出现两条红色条带,易混品及空白对照出现一条红的条带。琼脂糖凝胶电泳验证正确。对照药材克隆测序结果于蛤蚧mtDNA cytb基因同源性100%。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、灵敏度高于琼脂糖凝胶电泳10倍。结论PCR-免疫胶体金试纸条鉴定方法能够特异、准确、快速、可视化地鉴定蛤蚧的真伪,一体化快速检测试剂盒使鉴定方法更加规范化、标准化,更适合实地现场检测、适合普遍推广使用。

OBJECTIVE To establish a rapid molecular identification method for Chinese herbal medicine mealybug by PCR-immunocolloidal gold test strips and to develop a rapid gene detection kit.METHODS Screening methods and reagents for extracting genomic DNA.Designing species-specific primers and labeling according to mealybug mtDNA cytb gene,screening the best reaction conditions,developing PCR gene detection reagents,preparing control herbal DNA clones by molecular cloning and gene sequencing techniques,and using immunocolloidal gold test strips to detect the results.Further,an integrated rapid genetic assay kit is developed and evaluated for specificity,reproducibility,sensitivity and stability.RESULTS The integrity,concentration,and purity of genomic DNA met the requirements of PCR.The immunocolloidal gold test strips showed two red bands for mealybug control herb and authentic product,and one red band for easy mixed product and blank control when the primers were cycled 20 times at annealing temperature of 63℃.The agarose gel electrophoresis was verified to be corect.The clone sequencing result of the control herb was 100% homologous to mealybug mtDNA cytb gene.The integrated rapid gene detection kit showed high specificity,good reproducibility,stability and 10 times higher sensitivity than agarose gel electrophoresis.CONCLUSION The PCR-immune colloidal gold test strip identification method can specifically,accurately,quickly and visually identify the authenticity of mealybugs.The integrated rapid test kit makes the identification method more regulated and standardized,more suitable for field testing and suitable for universal promotion.