冬虫夏草PCR快速检测试剂盒研制与效果评价.docVIP

冬虫夏草PCR快速检测试剂盒研制与效果评价 　　[摘要]冬虫夏草为我国传统名贵中药材。资源短缺、价格昂贵和巨大的市场需求导致市售冬虫夏草掺伪掺假现象严重，亟需建立快速有效的鉴别方法。该研究基于实验前期已建立的冬虫夏草快速鉴定SSPCR方法，按照体外诊断试剂评价标准开发试剂盒，并对试剂盒的特异性、检测限、重复性及保存期4项性能进行了评价。结果显示冬虫夏草质量占混合物质量的1/200（质量分数05%以上）以上时可被成功检测。试剂盒对冬虫夏草及其伪品的检测结果显示，仅有冬虫夏草可被成功扩增，并在紫外下显示绿色荧光。该试剂盒37 ℃温育7 d仍有效，说明4 ℃保存其在1年内稳定有效。同一批次试剂盒在不同条件下，不同批次试剂盒在相同条件下，其检测结果均一致且准确，表明试剂盒具有良好的批内及批间重复性。将该试剂盒用于市售冬虫夏草及其混伪品的检测，操作简单快速，结果准确可靠，为下一步中药快检试剂盒的商品化奠定了基础。 　　[关键词]冬虫夏草； 聚合酶链式反应（PCR）； 检测试剂盒Development and evaluation of 　　[Abstract]Ophiocordyceps sinensis is a valuable traditional Chinese medicine. Due to resource shortage， expensive price and huge market demand， there are many adulterants of O. sinensis in markets. Therefore， it is necessary to establish a rapid and effective method for distinguishing O. sinensis. Based on the speciesspecific PCR of O. sinensis， this study developed a detection kit by optimizing the components and evaluated the specificity， detection limit， repeatability and shelf life of the kit. The results showed that when the quality of O. sinensis accounted for more than 1/200 of that mixture， it could be detected successfully. Moreover， only O. sinensis could be amplified and glowed bright green fluorescence under ultraviolet light. The kit was still in effect when it was placed at 37 ℃ for three days， which indicated that it was stable and effective for one year stored in 4 ℃. The kit in the same batch under different operation conditions， and in different batch under the same operation conditions gave the same result and accuracy， which showed good repeatability of the kit. It is simple， rapid and accurate to distinguish O. sinensis from its adulterants using the kit， and lays the foundation for commercialization of traditional Chinese medicine fast detection kit. 　　[Key words]Ophiocordyceps sinensis； PCR； detection kit 　　冬虫夏草是麦角菌科真菌冬虫夏草菌Ophiocordyceps sinensis寄生在蝙蝠蛾科昆虫幼虫上的子座和幼虫尸体的干燥复合体[1]，具有补肾益肺，止血化痰的功效，临床用于治疗肾虚精亏，久咳虚喘等症，为我国传统名贵中药材。由于野生?Y源有限且受到过度采挖，规模化的人工培育也尚未实现，导致冬虫夏草价格昂贵。巨大的市场需求也导致市售冬虫夏