基于MLPA结合熔解曲线法鉴别山药及其常见混伪品

Identification of Dioscoreae Rhizoma and Common Adulterants Based on MLPA Combined with Melting Curve Method

目的:利用MLPA技术结合熔解曲线法建立一种能鉴别山药及其常见混伪品的新方法。方法:以山药、参薯、木薯、番薯为研究对象,针对四个物种的matK序列分别设计MLPA特异性探针,通过DNA变性、杂交、连接、扩增及熔解反应等步骤,建立MLPA-熔解曲线鉴别体系,利用各样品熔解曲线中特异性峰(Tm值)的差异实现不同物种的鉴定。结果:27份样品中,山药Tm值为78.3℃,参薯Tm值为79.9℃,木薯Tm值为81.2℃,番薯Tm值为82.9℃,且相互之间没有交叉干扰。该法对山药、参薯、木薯、番薯各自单独的最低检测限为0.1 ng/μL;对相互混淆掺伪的最低有效检出率为5%。27份样品的检测结果均与测序结果一致。结论:MLPA技术结合熔解曲线法具有特异性强、灵敏度高、操作简单等优点,具有进一步推广应用的潜力。

Objective:To establish an identification method for Dioscoreae Rhizoma and its common adulterants by using MLPA technique and melting curve.Methods:With Dioscoreae Rhizoma and the tubers of Dioscorea alata,Manihot esculenta,Ipomoea batatas as the research object,MLPA-specific probes were designed for matK sequences of four species.MLPA-fusion curve identification system was established by DNA denaturation,hybridization,ligation,amplification and fusion reaction,respectively.The identification of different species was achieved by using the difference of specific peaks(Tm values)in the melting curves of each sample.Results:Among the 27 samples,the Tm value of Dioscoreae Rhizoma was 78.3℃,the Tm value of Dioscorea alata tubers was 79.97℃,the Tm value of Manihot esculenta tubers was 81.2℃and the Tm value of Ipomoea batatas tubers was 82.9℃,and there was no cross interference between each other.The lowest detection limit of Dioscoreae Rhizoma,and the tubers of Dioscorea alata,Manihot esculenta,Ipomoea batatas was 0.1 ng/μL.The minimum effective detection rate for mutual confusion was 5%.The results of 27 samples were consistent with the sequencing results.Conclusion:MLPA technology combined with melting curve method has the advantages of strong specificity,high sensitivity and simple operation,so it has the potential to be further popularized and applied.