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

Abstract

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 Mg2+ , 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 Mg2+ , 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. Key words: Anisakis simplex; Loop-mediated isothermal amplification; Identification