基于实时荧光定量PCR技术的当归不同炮制品中金黄色葡萄球菌含量测定方法的开发及比较  

Development and Comparison of Determination of Staphylococcus aureus in Different Processed Products of Angelicae Sinensis Radix Based on Real-time PCR Technique

作　　者：严维花 曹虹虹 郭爽 白德涛 陈杰 毛春芹[1] 李林[1] 陆兔林[1] YAN Wei-Hua;CAO Hong-hong;GUO Shuang;BAI De-tao;CHEN Jie;MAO Chun-qin;LI Lin;LU Tu-lin(College of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Gansu Zhongtian Pharmaceutical Co.Ltd.,Dingxi 743000,China)

机构地区：[1]南京中医药大学药学院,南京210023 [2]甘肃中天药业有限责任公司,甘肃定西743000

出　　处：《中国实验方剂学杂志》2020年第23期137-144,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家重点研发计划项目(2018YFC1707000,2017YFC1701902);第六批全国老中医药专家学术经验继承项目(国中医药人教发[2017]29号)。

摘　　要：目的:对当归不同炮制品中金黄色葡萄球菌的变化情况进行定量分析。方法:建立了实时荧光定量聚合酶链式反应法(Real-time PCR),对不同产地、不同收集企业、不同储藏时间当归饮片中金黄色葡萄球菌进行定量分析。荧光定量反应体系为SYBR Premix Ex Taq Ⅱ10μL,正向引物、反向引物(10μmol·L-1)各0.8μL,模板/基因组DNA 1μL,双蒸水7.4μL。荧光定量反应条件为①扩增曲线:94℃预变性30 s,94℃变性10 s,60℃退火12 s,72℃延伸30 s,循环45次,72℃单点检测信号;②熔解曲线:72℃开始检测,以0.5℃为台阶温度停留15 s采集荧光。根据Real-time PCR的测定结果,分别从生当归、酒当归和土炒当归中选择具有代表性的样品进行平板计数法测定,并与Real-time PCR检测结果进行比较。结果:不同炮制品中金黄色葡萄球菌含量排序为生当归>土炒当归>酒当归;在不同产地的当归饮片中均以甘肃渭源地区样品所含的金黄色葡萄球菌含量为最低;与零售企业相比,生产销售企业的生当归和酒当归中金黄色葡萄球菌含量较低;不同储藏时间对生当归和酒当归中金黄色葡萄球菌含量有一定的影响,随储藏时间的增加,金黄色葡萄球菌的含量增加。对部分代表性样品进行检测时发现,平板计数法检测结果数量级较Real-time PCR低3~4个数量级。结论:建立的Real-time PCR的特异性、灵敏度、准确性以及报告周期均优于平板计数法,可为当归不同炮制品中金黄色葡萄球菌的快速、准确定量检测提供有效技术手段。Objective: To quantitatively analyze the changes of Staphylococcus aureus in different processed products of Angelicae Sinensis Radix. Method: The real-time fluorescence quantitative polymerase chain reaction method(Real-time PCR)was established to quantitatively analyze S. aureus in Angelicae Sinensis Radix decoction pieces which bought from different producing areas,different enterprises and different storage time. The fluorescence quantitative reaction system was SYBR Premix Ex Taq Ⅱ of 10 μL,each of forward primer and reverse primer(10 μmol·L-1)of 0.8 μL,template/genome DNA of 1 μL,double distilled water of 7.4 μL. The reaction conditions of the fluorescence quantitative amplification curve were pre-denaturing for 30 s at 94 ℃,denaturing for 10 s at 94 ℃,annealing for 12 s at 60 ℃,extensing for 30 s at 72 ℃,cycling 45 times,single-point detection signal at 72 ℃. The melting curve was made from 72 ℃,and the step temperature of 0.5 ℃ was kept for 15 s to collect fluorescence. According to the results of Real-time PCR,representative samples were selected from Angelicae Sinensis Radix decoction pieces for comparison between plate counting method and Real-time PCR. Result:The content of S. aureus in different processed products was sorted by rank of raw Angelicae Sinensis Radix>soil-fried Angelicae Sinensis Radix>wine-processed Angelicae Sinensis Radix.The content of S. aureus was the lowest in the samples from Weiyuan area of Gansu province by comparing with other producing areas. Compared with the retail enterprises,the content of S. aureus in raw products and wineprocessed products from production and sale enterprises was lower. Different storage time had certain effect on the content of S. aureus in raw products and wine-processed products,and the content of S. aureus increased with the increase of storage time. The detection results of plate counting method were 3-4 orders of magnitude lower than that of Real-time PCR. Conclusion:The established Real-time PCR is superior to plate countin

关 键 词：当归饮片 炮制品 金黄色葡萄球菌 实时荧光定量聚合酶链式反应法(Real-time PCR) 致病菌 特异性引物 平板计数法 

分 类 号：R28[医药卫生—中药学] R943.1[医药卫生—中医学]