Development of a Real-Time PCR Assay for Detection of Kudoa iwatai (Myxosporea: Multivalvulida) in Japanese Seabass ( Lateolabrax japonicus)

Journal of Food Protection
Natsuko Iida-AoyamaKentaro Kawatsu

Abstract

Kudoa iwatai, a myxosporean parasite, has low host fish specificity, and consumers encounter commercial marine fish or marketed marine fish infected with this parasite in Japan. Although the presence of this parasite infection in fish samples is traditionally determined by the microscopic morphological examination of extracted spores, this method lacks sensitivity and specificity. In this study, we developed a real-time PCR assay for the detection of K. iwatai 18S rDNA to achieve the rapid and specific identification of K. iwatai in foreign substance inspection. We also evaluated the usefulness of real-time PCR for Japanese seabass ( Lateolabrax japonicus) with or without K. iwatai cysts. Our real-time PCR assay was able to reliably detect the target plasmid DNA over a 7-log range (from 4.0 × 101 to 4.0 × 107 copies per reaction) and displayed a linear relationship, with a correlation of determination value of 0.9993 and slope of -3.3651. Moreover, the mean value of the intra-assay coefficient of variation was 0.89% in triplicate assays, and the detection limit of this method was 2.5 copies of K. iwatai 18S rDNA per reaction. The sensitivity of the real-time PCR was the same or higher than that of an established conventional PC...Continue Reading

References

Jul 18, 2001·The Journal of Eukaryotic Microbiology·M L KentC Xiao
Jul 15, 2009·Molecular and Cellular Probes·Maeva RobertIsabelle Arzul
Jan 28, 2012·Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America·Takao KawaiTakahiro Ohnishi

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