| 龟甲的特异性PCR及高分辨率熔解曲线技术鉴别方法研究 |
| 投稿时间：2019-04-10 点此下载全文
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| 引用本文：张雪艳,袁媛,胡启跳,蒋超,方成武.龟甲的特异性PCR及高分辨率熔解曲线技术鉴别方法研究[J].中国现代中药,2019,21(9):1215-1220 |
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| 基金项目:中央本级重大增减支项目（2060302）；中央级公益性科研院所基本科研业务费专项（ZZ10-008） |
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| 中文摘要:目的：建立高效、稳定的龟甲Testudinis Carapax et Plastrum DNA鉴别方法。方法：利用龟甲的特异性聚合酶链式反应（Polymerase Chain Reaction，PCR）鉴别方法和高分辨率熔解曲线（HRM）鉴别方法，根据龟甲及其混伪品COI序列差异，寻找特异性单核苷酸多态性（single nucleotide polymorphism，SNP）位点设计鉴别引物，并对PCR反应条件进行优化；并使用通用引物扩增COI区域，产物直接进行HRM分析。结果：当退火温度为60 ℃，循环次数为35，使用设计的龟甲特异性鉴别引物GJ-360.F/R进行PCR扩增，经扩增均获得约360 bp特异性鉴别条带，混伪品皆无条带；使用通用引物RonM-tl和Vl-tl进行PCR扩增，产物使用HRM分析，龟甲药材在模板DNA质量浓度为3.5~87.9 ng·μL-1、引物浓度为0.2 μmol·L-1、镁离子浓度为2.0 mmol·L-1的条件下，龟甲药材标准熔解曲线为双峰，其Tm均值分别为（83.15±0.76）、（87.53±0.69）℃。结论：特异性PCR和HRM均可作为一种快速准确鉴别龟甲正伪品的鉴定方法。 |
| 中文关键词:龟甲 特异性聚合酶链式反应 分子鉴定 高分辨率熔解曲线 |
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| Identification of Testudinis Carapax et Plastrum by Specific PCR Method and High-resolution Melting Analysis |
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| Abstract:Objective：To establish an efficient and stable DNA identification method for Testudinis Carapax et Plastrum（TCP）.Methods：Based on the COI sequence differences between TCP and its adulterants，specific SNP loci were designed and primers were identified by using specific PCR and high-resolution melting curve（HRM）methods，and the conditions of PCR reaction were optimized.The COI region was amplified by universal primers，and the products were analyzed by HRM directly.Results：When the annealing temperature was 60 ℃ and the cycle number was 35，a specific band of 360 bp was visual on the gel electropherogram were using the designed TCP specific primer Guijia.F/R.high-resolution melting curve（HRM）technology was also applied to identification of TCP using COI universal primers.The annealing temperature was 60 ℃ and cycle number was 40.With chosen appropriate DNA template concentration，and the primer concentration as well as Mg2+concentration，an HRM model was established.The high melting curve of TCP was bimodal melting curve under these conditions，when the DNA template concentration was 3.5-87.9 ng·μL-1，the primer concentration was 0.2 μmol·L-1，Mg2+concentration was 2.0 mmol·L-1，the Tm value of HRM curve was（83.15±0.76）and（87.53±0.69），respectively.Conclusion：The results show that these two methods could use as rapid and accurate identification method for identify TCP and its adulterants. |
| keywords:Testudinis Carapax et Plastrum specific PCR molecular identification High-resolution melting curve |
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