关键词: 异尖线虫病, 异尖线虫, ITS?鄄2序列, 荧光定量PCR, SYBR Green Ⅰ

Abstract: 【Abstract】 Objective To establish an SYBR Green Ⅰ real-time quantitative PCR method for the detection of anisakid nematodes with zoonotic potential from Taiwan Strait. Methods Anisakid larvae of six species （Anisakis sim-plex, A. physeteris, Raphidascaris trichiuri, Contracaecum aduncum, C. muraenesoxi, and Contracaecum sp., a predominant species in fishes in the strait） were obtained from the guts of marine fishes and identified chiefly based on the morphol-ogical features. The ITS-2 rDNA sequences from the larvae were amplified by PCR using universal primers, then cloned and bidirectionally sequenced. According to these sequences, six specific forward primers were designed and synthesized. Specificity was determined by a series of conventional PCR respectively, the ITS-2 sequences amplified above were cloned into T vector which was subsequently transformed into E. coli DH5α. Following extraction and identification, the positive recombinant plasmid was used as quantitative template to generate standard curve and melt curve. Sensitivity and reprod-ucibility were determined. Results All the 6 standard curves established by the recombinant plasmids showed adequate linear relationship between threshold cycle（Ct） and template concentration. Melt curves were specific and all the 6 corr-elation coefficients were above 0.998. In the reproducibility test, the coefficients of variation （cv） of Ct values for detection of the 6 nematodes ranged between 0.18% and 2.80%, and the cv of the inter-assay ranged between 0.55% and 1.94%. The sensitivity of the real-time PCR was 1×102 copies/μl, about 100 times higher than the conventional PCR assays. The real-time quantitative PCR detection needed only 3.5 hours from the sample treatment to result report. Conclusion An SYBR Green Ⅰ fluorescent quantitative PCR has been developed for detecting anisakid nematodes with adequate sensitivity and specificity.

Key words: Anisakiasis, Anisakid nematode, ITS-2 sequence, Real-time PCR, SYBR Green Ⅰ