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为建立一种快速、准确的动物肉品中旋毛虫检测方法,根据旋毛虫ITS-Ⅱ基因序列,设计环介导等温扩增技术(loop-m ediated isothermal amplification,LAMP)引物,进行LAMP扩增。将旋毛虫DNA进行梯度稀释,以其为模板分别进行LAMP和常规PCR扩增,分析LAMP检测技术的敏感性。分别以旋毛虫、弓形虫、捻转血矛线虫、柔嫩艾美耳球虫、猪囊尾蚴、大肠杆菌等病原的基因组DNA为模板,进行LAMP检测,分析该检测方法的特异性。用该LAMP检测方法,分别对模拟样品和市售猪舌、鸭舌、鸡舌、羊舌和牛舌等110份样品进行旋毛虫感染情况检测。LAMP扩增产物的电泳结果和可视化SYBR G reen I荧光染料颜色的变化均表明该LAMP检测方法建立成功。当模板DNA稀释到10~(12)时亦可以检测,其敏感性是常规PCR的1 000倍。特异性研究发现,只有以旋毛虫基因组DNA为模板所进行的LAMP检测才出现阶梯形的电泳条带并且其SYBR Green I荧光染料呈现绿色荧光。在2 g肌肉中添加1条旋毛虫肌幼虫时亦能被该LAMP方法检测出来。但110份舌肌样品中均未检测到旋毛虫,LAMP检测结果与常规PCR结果一致。结果表明,本研究建立了基于旋毛虫ITS-Ⅱ基因的LAMP检测技术,该技术具有较好的敏感性和特异性,在样品检测中具有简便快捷的优点。
In order to establish a rapid and accurate method for detection of Trichinella in animal meat products, LAMP amplification was carried out based on the sequence of ITS-Ⅱ gene of loopworm and loop-m ediated isothermal amplification (LAMP) primers . The Trichinella spiralis DNA was gradient-diluted and used as templates for LAMP and routine PCR amplification respectively to analyze the sensitivity of LAMP detection technology. The specificity of this assay was analyzed by LAMP assay using genomic DNA of Trichinella spiralis, Toxoplasma gondii, Haemonchus contortus, Eimeria tenella, Cysticercus cellulosae and Escherichia coli as templates. The LAMP method was used to detect Trichinella spiralis infection in 110 samples including simulated tongue, tongue, tongue, tongue, tongue and tongue. The results of the electrophoresis of the LAMP amplification product and the visualization of the color of the SYBR Geenen fluorescent dye all indicate that the LAMP detection method is successfully established. When the template DNA is diluted to 10 ~ (12) can also be detected, the sensitivity of conventional PCR 1 000 times. Specific studies showed that only ladder-like electrophoresis bands appeared with LAMP assay using Trichinella genomic DNA as a template and their SYBR Green I fluorescent dyes showed green fluorescence. Adding 1 Trichinella muscle larva to 2 g of muscle can also be detected by this LAMP method. However, no Trichinella were detected in 110 samples of tongue muscle. The results of LAMP were consistent with those of conventional PCR. The results showed that LAMP detection technology based on Trichinella spiralis ITS-Ⅱ gene was established in this study. It has good sensitivity and specificity and has the advantages of simple and quick in the detection of samples.