鹿胎DNA提取方法、PCR参数优化及其快速检测试剂的开发应用

周亭亭, 艾金霞, 高丽君, 孙丽媛, 李明成, 张丽华

中国药学杂志 ›› 2019, Vol. 54 ›› Issue (18) : 1481-1485.

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中国药学杂志 ›› 2019, Vol. 54 ›› Issue (18) : 1481-1485. DOI: 10.11669/cpj.2019.18.005
·论著·

鹿胎DNA提取方法、PCR参数优化及其快速检测试剂的开发应用

  • 周亭亭1, 艾金霞1, 高丽君1, 孙丽媛1, 李明成1,2*, 张丽华3
作者信息 +

Development and Application of DNA Extraction Methods, PCR Parameter Optimization and Rapid Detection Reagent for Fetus Cervi

  • ZHOU Ting-ting1, AI Jin-xia1, GAO Li-jun1, SUN Li-yuan1, LI Ming-cheng1,2*, ZHANG Li-hua3
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文章历史 +

摘要

目的 优化鹿胎DNA提取方法、聚合酶链式反应(PCR)反应参数,建立一种特异、准确的鹿胎快速检测试剂。方法 采用改良盐析法、改良十二烷基硫酸钠(SDS)法A、B提取鹿胎DNA;选取市面常见4种DNA聚合酶进行对比研究;运用经典的控制变量法和交叉实验优化退火温度及退火时间;建立鹿胎快速检测试剂,并对其进行评价。结果 采用改良SDS法B提取的DNA,其A260/A280比值为(1.74±0.05),质量浓度为(0.250±0.005)μg·L-1;选用高保真Taq DNA聚合酶,其PCR产物浓度可达(0.185±0.005)μg·L-1;筛选出最适退火温度58 ℃和退火时间30 s;建立的快速检测试剂能够特异准确鉴定鹿胎及其伪品,且在563 bp处有1条清晰明亮条带。结论 鹿胎快速检测试剂融合了最优DNA提取方法、最优PCR反应参数,能够高准确性、高特异性完成鹿胎及其伪品的鉴定工作。

Abstract

OBJECTIVE To optimize DNA extraction methods and PCR reaction parameters, and develop an excellent and accurate rapid detection reagent for Fetus Cervi. METHODS The DNA of Fetus Cervi was extracted by the modified salting method, modified SDS method A and modified SDS method B. Four DNA polymerase were chosen from the market and compared with each other. The annealing temperature and annealing time were optimized by classical control variable method and intersected experiment. A rapid detection reagent of Fetus Cervi was developed and then evaluated. RESULTS The A260/A280 ratio of the DNA extracted by modified SDS method B was (1.74±0.05), and the mass concentration was (0.250±0.005)μg·L-1. With high fidelity Taq DNA polymerase, the PCR product concentration could reach (0.185±0.005)μg·L-1. Through these experiments, the annealing temperature was set at 58 ℃ and the annealing time was 30 s. The rapid detection reagent course was established to quickly and accurately identify Fetus Cervi and their artefacts, with one clear and bright band at 563 bp. CONCLUSION The rapid detection reagent of Fetus Cervi combines the optimal DNA extraction method and the optimal PCR reaction parameters, and can accomplish the identification of Fetus Cervi and its pseudo-products with high accuracy and specificity.

关键词

鹿胎 / DNA提取优化 / 退火温度 / 快速检测

Key words

Fetus Cervi / DNA extraction optimization / annealing temperature / rapid detection

引用本文

导出引用
周亭亭, 艾金霞, 高丽君, 孙丽媛, 李明成, 张丽华. 鹿胎DNA提取方法、PCR参数优化及其快速检测试剂的开发应用[J]. 中国药学杂志, 2019, 54(18): 1481-1485 https://doi.org/10.11669/cpj.2019.18.005
ZHOU Ting-ting, AI Jin-xia, GAO Li-jun, SUN Li-yuan, LI Ming-cheng, ZHANG Li-hua. Development and Application of DNA Extraction Methods, PCR Parameter Optimization and Rapid Detection Reagent for Fetus Cervi[J]. Chinese Pharmaceutical Journal, 2019, 54(18): 1481-1485 https://doi.org/10.11669/cpj.2019.18.005
中图分类号: R284   

参考文献

[1]AI J X, LI M C, XIA W, et al. Application of mitochondrial DNA in the identification of Fetus Cervi[J]. Chin Pharm J(中国药学杂志), 2017, 52(18):1589-1593.
[2]YANG C, LI J, XU Y, et al. Extraction and comparison of pork, beef and mutton DNA by 4 methods[J]. Food Eng(食品工程), 2018(3):51-53,61.
[3]DENG Y, LI M C, ZHANG L H. Identification of the characteristics of the genuine and pseudo-products of tortoise by compound polymerase chain reaction[J]. Chin Pharm J(中国药学杂志), 2014, 49(12):1022-1026.
[4]LI T H, WANG J, LI M C, et al. Study on the specificity identification of cytochrome b gene of Chinese herbal medicine tortoise[J]. Chin Pharm J(中国药学杂志), 2012, 47(3):182-185.
[5]ZHOU T T, YU W J, LI M C, et al. Development and evaluation of unibract fritillary bulb DNA detection kit[J]. Chin Pharm J(中国药学杂志), 2014, 49(6):501-504.

基金

吉林省中医药科技项目资助(2017087);吉林省科技发展计划项目资助( 20160204004NY);吉林省科技发展计划项目资助(20170307001YY);吉林省级产业创新专项资金项目资助(2018C048-2);吉林省教育厅“十三五”产业化项目资助(JJKH20180377KJ);吉林省科技发展计划项目资助(20180201023YY);吉林省科技发展计划资助项目资助( 20160307030YY);吉林省科技发展计划项目资助(2019031014NY);吉林省发展计划项目资助(20140307008YY);吉林省科技创新中心建设项目(20190902018TC)
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