中华鲟TaqMan实时荧光PCR检测方法的建立

摘要

为给中华鲟提供及时而有效的保护,本研究根据NCBI上已公布的中华鲟D-loop基因序列设计并筛选了引物与探针组合,建立了中华鲟TaqMan实时荧光PCR检测方法,并对所建立的实时荧光PCR方法进行方法学评价.结果显示,建立的方法能够对中华鲟及其产品进行快速而准确的检测,且特异性好,检测灵敏度高,能够达到0.001 ng的中华鲟DNA和100个拷贝的重组质粒DNA.该方法在应用于全国多个地区采集的鲟鱼样品的检测时,结果表明只有7个从相关科研单位得到的已经鉴定的中华鲟标本,经检测才是真正的中华鲟,而许多餐馆中号称的"中华鲟"经检测并非真正的中华鲟,只是其他的一些鲟鱼品种.因此本研究建立的中华鲟TaqMan实时荧光PCR检测方法特异性强、灵敏度高,且具有较好的应用价值.%To provide timely and effective protection to Acipenser sinensis,a combination of primer and probe was de-signed and filtered according to D-loop sequence published by NCBI,and a detection method of TaqMan real-time PCR for Acipenser sinensis was established and evaluated in this study. The results showed that the method could be used to detect Acipenser sinensis and its products,with high specificity and high sensitivity,and its detection sensitivity could reach 0.001 ng DNA of Acipenser sinensis and 100 copies of the recombinant plasmid DNA.When this method was applied in detection of some Acipenser sinensis samples across the country,only seven Acipenser sinensis specimens collected from relevant research institutes had been confirmed to be real,and some commercially so-called Acipenser sinensis in many restaurants were in fact other sturgeon species. Therefore,the detection method of TaqMan real-time PCR for Acipenser sinensis has high specificity, high sensitivity and great significance in application.