简单异尖线虫环介导等温扩增（LAMP）鉴定方法的建立和应用

The establishment of loop-mediated isothermal amplification （LAMP） for identification of Anisakis simplex

目的 建立快速鉴定简单异尖线虫的环介导等温扩增方法（LAMP）. 方法 根据简单异尖线虫ITS保守区域的基因序列设计特异性引物进行LAMP扩增,对反应体系中dNTPs、Mg^2＋、Betaine和2对引物的浓度,以及反应温度、反应时间等进行优化;在优化后的体系下,将10倍比稀释的简单异尖线虫ITS序列重组质粒进行灵敏性试验;用典型异尖线虫、对盲囊线虫、针蛔线虫对该方法进行特异性试验;并用7份简单异尖线虫样品进行验证. 结果 LAMP的最佳反应体系为0.8 mmoL/L dNTPs、10 mmol/L Mg^2＋、0.8 mmoL/L Betaine、0.2μmol/L外引物、1.6μmoL/L内引物、8 U Bst DNA聚合酶大片段以及适量的模板,反应温度为62℃,反应时间为60 min;检测简单异尖线虫的灵敏度达到10拷贝/μl,且对典型异尖线虫、对盲囊线虫、针蛔线虫无交叉反应;7份简单异尖线虫样品经LAMP验证均为阳性.结论 LAMP方法灵敏性高、特异性强,并且操作简便、成本低,是鉴定简单异尖线虫的有效手段.

Objective To establish a simple and quick method of loop-mediated isothermal amplification （LAMP） for identification of Anisakis simplex. Methods According to the ITS gene sequence of A. simplex , a set of specific primers was designed to amplify the specific DNA sequence by LAMP. The concentrations of Mg^2＋ , Betaine, dNTPs and primers were optimized. The sensitivity and specificity of the method were also tested. Then 7 samples of A. simplex were validated by LAMP. Results The optimal reaction system contained 0. 8 mmol/L dNTPs, 10 mmol/L Mg^2＋ , 0. 8 mmol/L Betaine, 0. 2 （μmol/L exogenous primers, 1.6 μmol/L inner primers and 8 U Bst DNA polymerase. The optimal reaction temperature was 62 ℃ and reaction time was 60 min. Under those conditions, the detection limit for A. simplex DNA template was 10 copies/μl and no cross reaction with A. typica, Contracaecum sp. , Raphidascaris trichiuri was found. All of the samples of A. simplex were positive by LAMP. Conclusion The LAMP is a sensitive, specific, economic and effective method to identify A. simplex.