[Detection of anisakid nematodes by an SYBR green I real-time PCR]

To establish an SYBR Green I real-time quantitative PCR method for the detection of anisakid nematodes with zoonotic potential from Taiwan Strait.

Anisakid larvae of six species (Anisakis simplex, A. physeteris, Raphidascaris trichiura, 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 morphological 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 DH5alpha. Following extraction and identification, the positive recombinant plasmid was used as quantitative template to generate standard curve and melt curve. Sensitivity and reproducibility were determined.

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 correlation 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 x 10(2) copies/microl, 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.

An SYBR Green I fluorescent quantitative PCR has been developed for detecting anisakid nematodes with adequate sensitivity and specificity.