Establishment and Application of a Real-time Fluorescence Loop-mediated Isothermal Amplification Method for the Detection of Anisakis typical in Imported Aquatic Products
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Abstract:
The present study established a real-time fluorescence loop-mediated isothermal amplification (LAMP) method for detecting the Anisakis typical in imported aquatic products. According to the mechanism of LAMP, three sets of specific primers for targeting the ITS2 rDNA were designed for specific identification of target genes . The specificity of primers were tested among Anisakis typical, Anisakis simplex, Anisakis brevispiculata, Anisakis physeteris, Anisakis nascettii, Hysterothylacium Spp., Contracaccum Spp. and Gnathostoma spp.. The sensitivity of this LAMP method was tested with the template concentrations ranging from 1 ng/μL to 10 ag/μL, which was 100 times higher than the traditional PCR method. Besides, 15 times repeats were conducted at the detection limit of 1 fg/μL plasmid template. This LAMP method was used to detect Anisakis typical in 41 samples imported from different tropic countries and areas, and its false positive rate was zero compared with the conventional PCR method. The established real-time fluorescence LAMP method is suitable for specific A. typical detection in imported aquatic products.
