Identification of Dioscoreae Rhizoma and Common Adulterants Based on MLPA Combined with Melting Curve Method
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 sys-tem was established by DNA denaturation,hybridization,ligation,amplification and fusion reaction,respectively.The identification of dif-ferent 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.
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