| As one of the most rare Chinese medicinal materials,Tokay Gecko,the dry body of Gekko gecko Linnaeus,is well known for its remarkably therapeutic effect on cough,asthma and kidney deficiency.Due to the increasing market demand,declining supply,and phenomena of homonym and synonym,a considerable amount of adulterants have emerged on markets.Thus,it is necessary to establish an accurate and rapid identification method for distinguishing Gekko gecko from its adulterants and pinpointing it from highly processed products.In this study,to optimize the efficiency of DNA extraction for Gekko gecko.DNA extraction reagents and rinsing reagents were optimized.The results showed that the optimized method was applicable to the DNA extraction even for 1 mg of Gekko gecko.Two DNA extraction methods,the optimized method and DNA extraction kit,were compared.For the optimized method,these DNA concentrations of 1 mg and 30 mg of Gekko gecko were 2.72 ng/?L and 139.0 ng/?L,respectively.Meanwhile,1.63 ng/?L and 98.9 ng/?L for DNA extraction kit.Therefore,the optimized method in this study is more efficient,and it is conductive to the molecular identification for Gekko gecko.To establish a specific minibarcoding method for the rapid identification of Gekko gecko.In this study,293 samples of twenty species of Gekko gecko and adulterants,and fifteen products were collected.The total genomic DNA was extracted to amplify COI region.All the sequences were analyzed by using CodonCode Aligner V 3.0 and MEGA 6.0.Fifteen products were detected with the specific primers designed based on COI sequences.Results showed that the minimum inter-specific distance of Gekko gecko(0.262)was higher than the maximum intra-specific distance(0.148).Thirty-four haplotypes of Gekko gecko formed into a branch with the bootstrap value of 99%.It indicated that Gekko gecko could be effectively differentiated from its adulterants through intra-and inter-specific distances analysis,and NJ tree based on COI sequences.Two pairs specific primers were designed in this study.And specific primers COISF2/COISR2 and COISF3/COISR3 amplified 200 bp and 133 bp fragments of COI region respectively,which were both suitable for the identification of Gekko gecko and its adulterants.Furthermore,for COISF3/COISR3,the required minimal template concentration was less than 10 pg/?L,and it detected Gekko gecko from fifteen products.Therefore,the specific DNA minibarcoding method developed in this study is a powerful tool for the identification of Gekko gecko and its counterfeits,and it also can be applied to detect Gekko gecko from its highly processed products.To identify Gekko gecko and its adulteration efficiently,a method was developed for Gekko gecko,six common counterfeits,and ten commercial Gekko gecko powders through DNA barcoding combined with high resolution melting analysis(Bar-HRM)based on 12S rRNA regions.Specific primers were designed targeting 16S rRNA sequences of Gekko gecko and fourteen adulterants to amplify a minibarcode.Ten commercial Gekko gecko powders and fifteen products were detected based on the minibarcode.The results demonstrated that Gekko gecko and six common counterfeits could be clearly distinguished through Bar-HRM analysis based on 12S rRNA regions.The sensitivity of 12S-HRM in adulterant detection was 1%.The specific primers 16SSF2/16SSR2 designed for this study were suitable for the amplification of mini 16S rRNA regions(132 bp).Gekko gecko from commercial products were detected by using this Bar-HRM method based on mini 16S rRNA regions(132 bp).Overall,the Bar-HRM method with mini 16S rRNA barcode is a powerful tool for the identification of Gekko gecko and its adulterants.It is also practicable for detecting adulteration and highly processed products.In conclusion,Bar-HRM,the rapid identification technique,is applicable for the identification of Gekko gecko and its adulterants.This efficient method has a promising future for the detecting of highly processed products and other traditional Chinese medicine. |
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