利用分子方法鉴别白花蛇舌草及其伪品伞房花耳草

Identification of Hedyotis diffusa and its adulterant Hedyotis corymbosa by molecular method

目的:建立一种鉴别白花蛇舌草及其常见伪品伞房花耳草的分子方法,确保临床用药安全。方法:根据白花蛇舌草和伞房花耳草内转录间隔区2(ITS2)序列的差异设计特异探针,采用多重连接探针扩增(MLPA)技术和实时荧光定量PCR(real-time quantitative PCR,qPCR)技术,建立了一种能够鉴别白花蛇舌草、伞房花耳草的多重连接探针扩增-实时荧光定量PCR(MLPA-qPCR)方法。结果:对扩增产物进行熔解曲线分析,结果显示,所设计的MLPA探针具有良好的特异性,探针之间不存在交叉反应,白花蛇舌草探针的特异性熔解曲线T_(m)值为(81.8±0.2)℃,伞房花耳草探针的熔解曲线T_(m)值为(84.0±0.2)℃;灵敏度分析结果表明,该方法检出白花蛇舌草和伞房花耳草的最低模板量均为0.1 ng;掺伪检测试验结果表明,当白花蛇舌草探针与伞房花耳草探针比例为1.0∶0.6时,白花蛇舌草中掺杂1%的伞房花耳草仍可被有效检出。对收集的39份样品进行检测,成功鉴别白花蛇舌草25份、伞房花耳草12份、白花蛇舌草与伞房花耳草混合样品2份。结论:本研究所建立的MLPA-qPCR方法特异性强,灵敏度高,可作为鉴别白花蛇舌草及其常见伪品伞房花耳草的特异性鉴别方法。

Objective:To establish a molecular method for identification of Hedyotis diffusa and its common adulterant Hedyotis corymbosa,to ensure the safety of clinical drug use.Methods:Species-specific probes of Hedyotis diffusa and Hedyotis corymbosa were designed based on the difference of intergenic region 2(ITS2).Using multiplex ligation-dependent probe amplification(MLPA)and real-time quantitative PCR(qPCR),a MLPA-qPCR method was established to identify Hedyotis diffusa and Hedyotis corymbosa.Results:The melting curve analysis of the amplification products showed that the MLPA probes were designed with good specificity and there was no cross-reaction between the probes.The specific melting curve T_(m)value of Hedyotis diffusa probe was(81.8±0.2)℃,and that of Hedyotis corymbosa probe was(84.0±0.2)℃.Sensitivity test results showed that the sensitivity for detecting both components reached 0.1 ng.And the adulteration test showed that the Hedyotis diffusa with 1%Hedyotis corymbosa could still be detected effectively when the ratio of Hedyotis diffusa probe to Hedyotis corymbosa probe was 1.0∶0.6.39 samples collected were tested,and 25 samples of Hedyotis diffusa,12 samples of Hedyotis corymbosa and 2 mixed samples were successfully identified.Conclusion:The established MLPA-qPCR method with high specificity and sensitivity can be used for the specific identification of Hedyotis diffusa and its common adulterant Hedyotis corymbosa.