基于重组酶介导扩增技术的水牛角快速现场鉴别方法
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摘要
目的:以水牛角鉴别为例建立中药重组酶介导扩增(Recombination-aid amplification,RAA)快速鉴别方法,用于水牛角及其混伪品牦牛角的鉴别。方法:通过比较水牛角及其混伪品牦牛角基原物种的线粒体基因组序列差异,寻找高变异区域,根据变异位点设计水牛角正品水牛的特异性RAA鉴别引物SNJ-1.F和SNJ-1.R及荧光探针SNJ-1.probe,使用碱裂解法提取DNA,优化RAA反应体系,在37℃下恒温孵育15～20 min,通过凝胶电泳及实时荧光监测反应结果,使用DNA测序方法验证RAA鉴别结果的准确性。结果:在37℃恒温孵育20 min后,水牛角RAA产物凝胶电泳图谱出现约140 bp特异性鉴别条带,使用实时荧光RAA鉴别时,水牛角正品均在6.21～8.37 min出现特异性扩增,牦牛角样品均在10.08 min后才出现扩增,COI片段DNA测序鉴别结果与荧光RAA鉴别结果相吻合。结论:特异性RAA技术可在20 min内快速鉴别水牛角,可用于水牛角药材、饮片及其制品的快速现场鉴定。该方法具有简单、快速、无需大型仪器等特点,在中药现场鉴定、药检抽查等方面有着广阔的应用前景。
Objective:To establish a rapid on-site method for identifying Chinese medical material recombinase-mediated amplification(RAA) technology for the use of identifying medicinal Bubali Cornu from yak horn.Method:Based on the differences of mitochondrial genome sequences between Bubali Cornu and adulterants,the specific RAA primer(SNJ-1.F,SNJ-1.R) and fluorescence probe SNJ-1.probe were designed by variation sites.Alkaline lysis method was used to extract DNA from milled samples,and optimize RAA reaction system.The incubation was made at 37 ℃ for 15-20 min,the reaction results were monitored through gel electrophoresis and a mobile fluorescence amplification instrument.The RAA identification result was compared with COI DNA sequencing.Result:After incubation at 37 ℃ for 20 min,about 140 bp of bright and simple bands was amplified from DNA templates of Bubalus bubalis,whereas Bos mutus were negative.By the Real-time fluorescent RAA identify method,all reactions in DNAs from Bubali Cornu samples were amplified from 6.21 to8.37 min,whereas DNAs from yak samples were amplified after 10.08 min,COI sequencing results conformed to the Real-time fluorescent RAA identification.Conclusion:Specific RAA could rapidly identify Bubali Cornu in 20 minutes,and thus be applied in medical material and its products.This method is simple,rapid and sophisticated instrument-free,with promises in on-site identification of traditional Chinese medicine.
Objective:To establish a rapid on-site method for identifying Chinese medical material recombinase-mediated amplification(RAA) technology for the use of identifying medicinal Bubali Cornu from yak horn.Method:Based on the differences of mitochondrial genome sequences between Bubali Cornu and adulterants,the specific RAA primer(SNJ-1.F,SNJ-1.R) and fluorescence probe SNJ-1.probe were designed by variation sites.Alkaline lysis method was used to extract DNA from milled samples,and optimize RAA reaction system.The incubation was made at 37 ℃ for 15-20 min,the reaction results were monitored through gel electrophoresis and a mobile fluorescence amplification instrument.The RAA identification result was compared with COI DNA sequencing.Result:After incubation at 37 ℃ for 20 min,about 140 bp of bright and simple bands was amplified from DNA templates of Bubalus bubalis,whereas Bos mutus were negative.By the Real-time fluorescent RAA identify method,all reactions in DNAs from Bubali Cornu samples were amplified from 6.21 to8.37 min,whereas DNAs from yak samples were amplified after 10.08 min,COI sequencing results conformed to the Real-time fluorescent RAA identification.Conclusion:Specific RAA could rapidly identify Bubali Cornu in 20 minutes,and thus be applied in medical material and its products.This method is simple,rapid and sophisticated instrument-free,with promises in on-site identification of traditional Chinese medicine.
引文
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[3]杨璐,吴文如,付菲,等.新型快速中药材DNA提取方法的探索与应用[J].中草药,2019,50(2):502-509.
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[8]呙军.环介导等温扩增对何首乌及其混伪品的快速鉴定[D].广州:华南理工大学,2009.
[9]赵源,宋云,李明福,等.基于LAMP的人参物种鉴定技术的研究[J].植物检疫,2015,29(5):30-34.
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[11]刘旭朝,周丽思,刘金欣,等.基于COI序列的水牛角及其易混伪品DNA条形码鉴定研究[J].药学学报,2017,52(3):494-499.
[12]黄璐琦,袁媛.中药分子鉴定操作指南[M].上海:上海科学技术出版社,2014:49.
[2]蒋超,黄璐琦,袁媛,等.使用碱裂解法快速提取药材DNA方法的研究[J].药物分析杂志,2013,33(7):1081-1090.
[3]杨璐,吴文如,付菲,等.新型快速中药材DNA提取方法的探索与应用[J].中草药,2019,50(2):502-509.
[4]Notomi T,Okayama H,Masubuchi H,et al.Loopmediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):e63.
[5]Vincent M,XU Y,KONG H.Helicase-dependent isothermal DNA amplification[J].EMBO Rep,2004,5(8):795-800.
[6]Piepenburg O,Williams C H,Stemple D L,et al.DNAdetection using recombination proteins[J].PLo S Biol,2006,4(7):e204.
[7]TIAN E,LIU Q,YE H,et al.A DNA barcode-based RPA assay(BAR-RPA)for rapid identification of the dry root of Ficus hirta(Wuzhimaotao)[J].Molecules,2017,22(12):2261.
[8]呙军.环介导等温扩增对何首乌及其混伪品的快速鉴定[D].广州:华南理工大学,2009.
[9]赵源,宋云,李明福,等.基于LAMP的人参物种鉴定技术的研究[J].植物检疫,2015,29(5):30-34.
[10]国家药典委员会.中华人民共和国药典.一部[M].北京:中国医药科技出版社,2015:83.
[11]刘旭朝,周丽思,刘金欣,等.基于COI序列的水牛角及其易混伪品DNA条形码鉴定研究[J].药学学报,2017,52(3):494-499.
[12]黄璐琦,袁媛.中药分子鉴定操作指南[M].上海:上海科学技术出版社,2014:49.