基于DNA条形码、特异性引物鉴定益母草及其易混淆品

孟祥松; 王雨艨; 王孟虎; 郝婷婷; 孙一帆; 李啸云; 金阳

doi:10.13748/j.cnki.issn1007-7693.20232893

基于DNA条形码、特异性引物鉴定益母草及其易混淆品

Identification of Leonurus Japonicus and Its Confounding Products Based on DNA Barcoding and Specific Primers

摘要

摘要:
目的利用DNA条形码、特异性引物对益母草、夏至草进行鉴定。
方法基于ITS2通用引物对益母草、夏至草进行扩增并测序，构建NJ系统发育树；根据ITS2序列设计益母草、夏至草特异性引物，并对引物的特异性、退火温度、引物量、模板量、循环次数、检测限进行考察；基于特异性引物结合SYBR荧光定量PCR法检测益母草、夏至草的熔解温度(melting temperature，T_m)值，并对其检测限进行考察；基于普通PCR及荧光定量PCR分别对收集的样品真伪进行检测。
结果基于ITS2构建的NJ树显示益母草、夏至草均独立聚为1支；益母草、夏至草的引物均具有特异性，其最佳退火温度、引物量、模板量、循环次数分别为65.2 ℃、1.0 μL(10 mmol·L⁻¹)、80 ng、35次循环，普通PCR益母草、夏至草检测限分别达到4.0、40.0 ng·μL⁻¹。基于特异性引物结合SYBR荧光定量PCR可知：益母草、夏至草的T_m值分别为85.5~86.0 ℃、82.0~82.5 ℃，其检测限均达到1 pg·μL⁻¹。
结论基于DNA条形码、特异性引物、荧光定量PCR法均能够对益母草、夏至草进行鉴定，基于SYBR荧光定量PCR检测限远小于普通PCR的检测限，为益母草、夏至草微量掺伪鉴定提供依据。

Abstract:
OBJECTIVE To identify Leonurus japonicus and Lagopsis supina by DNA barcoding and specific primers.
METHODS The NJ phylogenetic tree was constructed by amplification and sequencing of L. japonicus and L. supina based on ITS2 universal primes. Specific primers of L. japonicus and L. supina were designed according to ITS2 sequence, and their specificity, annealing temperature, amount of primer, amount of template, number of cycles and detection limit were investigated. The melting temperature(T_m) values of L. japonicus and L. supina were detected by SYBR green fluorescent quantitative PCR based on specific primers, and their detection limits were investigated. Authenticity of the collected samples was detected by conventional PCR and qPCR respectively.
RESULTS The NJ tree based on ITS2 showned that L. japonicus and L. supina were clustered together independently. The primers of L. japonicus and L. supina were all specific. The optimal annealing temperature, primer volume, template volume and cycle times were 65.2 ℃, 1.0 μL(10 mmol·L⁻¹), 80 ng and 35 cycles, respectively. The detection limits of L. japonicus and L. supina were 4.0 ng·μL⁻¹ and 40.0 ng·μL⁻¹, respectively. The T_m values of L. japonicus and L. supina were 85.5−86.0 ℃ and 82.0−82.5 ℃, respectively, and their detection limits reached 1 pg·μL⁻¹ based on specific primes combined with SYBR green fluorescent quantitative PCR.
CONCLUSION L. japonicus and L. supina can be distinguished by DNA barcode, specific primer and quantitative fluorescence PCR. The detection limit of SYBR green fluorescent quantitative PCR is much lower than that of ordinary PCR. This study provides a basis for the identification of L. japonicus and L. supina.

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