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Molecular identification and real-time quantitative PCR (qPCR) for rapid detection of Thelohanellus kitauei, a Myxozoan parasite causing intestinal giant cystic disease in the Israel carp.

Seo JS, Jeon EJ, Kim MS, Woo SH, Kim JD, Jung SH, Park MA, Jee BY, Kim JW, Kim YC, Lee EH - Korean J. Parasitol. (2012)

Bottom Line: Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations.In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans.Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.

View Article: PubMed Central - PubMed

Affiliation: Pathology Division, National Fisheries Research and Development Institute, Busan 619-705, Korea.

ABSTRACT
Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.

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Linear standard curves derived from 10-fold serially diluted pKitauei plasmid (A) and DNA purified using known numbers of T. kitauei spores (B). x-axis and y-axis represent a log10 of number of spore used in DNA preparation and mean (± SD) real-time qPCR Cp value, respectively. PCR efficiency calculated using slope of the standard curve was 99.2%.
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Figure 3: Linear standard curves derived from 10-fold serially diluted pKitauei plasmid (A) and DNA purified using known numbers of T. kitauei spores (B). x-axis and y-axis represent a log10 of number of spore used in DNA preparation and mean (± SD) real-time qPCR Cp value, respectively. PCR efficiency calculated using slope of the standard curve was 99.2%.

Mentions: In this study, for early detection of the infection status, the detection limits of parasites were determined by PCR and real-time qPCR. PCR was carried out using various concentrations of the full-length 18S rRNA gene-harboring plasmid (pKitauei), the suspension of spores, genomic DNA from isolated spores, and genomic DNA from the spore-spiked tissues as templates. We initially carried out conventional PCR and real-time qPCR using different concentrations of the pKitauei plasmid for further use to estimate the DNA amount of T. kitauei. Conventional PCR was capable of detecting 10-3 pg of the pKitauei plasmid within only 30 cycles (Fig. 2A) and 10-4 pg of plasmid DNA could be detected over 35 cycles of real-time qPCR (Table 1). Linear standard curves could be generated by real-time qPCR from the Cq values of the diluted plasmid pKitauei and DNA purified from various numbers of spores (Fig. 3) and primer specificity was confirmed via melt-curve analysis in all samples (data not shown).


Molecular identification and real-time quantitative PCR (qPCR) for rapid detection of Thelohanellus kitauei, a Myxozoan parasite causing intestinal giant cystic disease in the Israel carp.

Seo JS, Jeon EJ, Kim MS, Woo SH, Kim JD, Jung SH, Park MA, Jee BY, Kim JW, Kim YC, Lee EH - Korean J. Parasitol. (2012)

Linear standard curves derived from 10-fold serially diluted pKitauei plasmid (A) and DNA purified using known numbers of T. kitauei spores (B). x-axis and y-axis represent a log10 of number of spore used in DNA preparation and mean (± SD) real-time qPCR Cp value, respectively. PCR efficiency calculated using slope of the standard curve was 99.2%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3375447&req=5

Figure 3: Linear standard curves derived from 10-fold serially diluted pKitauei plasmid (A) and DNA purified using known numbers of T. kitauei spores (B). x-axis and y-axis represent a log10 of number of spore used in DNA preparation and mean (± SD) real-time qPCR Cp value, respectively. PCR efficiency calculated using slope of the standard curve was 99.2%.
Mentions: In this study, for early detection of the infection status, the detection limits of parasites were determined by PCR and real-time qPCR. PCR was carried out using various concentrations of the full-length 18S rRNA gene-harboring plasmid (pKitauei), the suspension of spores, genomic DNA from isolated spores, and genomic DNA from the spore-spiked tissues as templates. We initially carried out conventional PCR and real-time qPCR using different concentrations of the pKitauei plasmid for further use to estimate the DNA amount of T. kitauei. Conventional PCR was capable of detecting 10-3 pg of the pKitauei plasmid within only 30 cycles (Fig. 2A) and 10-4 pg of plasmid DNA could be detected over 35 cycles of real-time qPCR (Table 1). Linear standard curves could be generated by real-time qPCR from the Cq values of the diluted plasmid pKitauei and DNA purified from various numbers of spores (Fig. 3) and primer specificity was confirmed via melt-curve analysis in all samples (data not shown).

Bottom Line: Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations.In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans.Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.

View Article: PubMed Central - PubMed

Affiliation: Pathology Division, National Fisheries Research and Development Institute, Busan 619-705, Korea.

ABSTRACT
Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.

Show MeSH
Related in: MedlinePlus