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PHD : Parasites, Hosts and Diseases

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Original Article

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

The Korean Journal of Parasitology 2012;50(2):103-111.
Published online: May 24, 2012

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Corresponding author (ehlee1234@nfrdi.go.kr)
• Received: November 23, 2011   • Revised: January 8, 2012   • Accepted: January 23, 2012

© 2012, Korean Society for Parasitology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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
Korean J Parasitol. 2012;50(2):103-111.   Published online May 24, 2012
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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
Korean J Parasitol. 2012;50(2):103-111.   Published online May 24, 2012
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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
Image Image Image
Fig. 1 Characterization of spores. (A) Various sizes of giant cysts (arrows) containing mature spores found in the intestine of an Israel carp infected with Thelohanellus kitauei. (B) Spores of T. kitauei isolated from the intestine of a diseased fish. Scale bar: 20 µm.
Fig. 2 Determination of the detection limit of conventional PCR using AccuPower™ PCR PreMix system. Four µl of PCR products were loaded on 1% agarose gels and ethidium bromide-stained specific PCR bands were visualized using a UV-transilluminator. PCR was carried out using various concentrations of the full-length 18S rRNA gene-harboring plasmid pKitauei (A), the suspension containing different number of spores (B), genomic DNA purified from isolated spores (C) and genomic DNA from the various numbers of spore-spiked tissues (D) as templates. M: 100 bp ladder from Bioneer.co.kr
Fig. 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%.
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
Samples Amount per reaction Mean Cq (SD) Samples Amount per reaction (no. of spores) Mean Cq (SD) Samples Amount per reaction (no. of spores) Mean Cq (SD) pKitauei 103 pg 9.8 (0.158) DNA from spore- spiked tissues 100 32.16 (0.643) Genomic DNA from isolated spores 5,000 21.75 (0.200) 102 pg 14.89 (0.290) 10 35.61 (0.946) 1,000 23.54 (0.147) 101 pg 18.79 (0.016) 1 38.04 (0.305) 500 24.27 (0.116) 100 pg 23.14 (0.040) 0.1 N/A 100 27.18 (0.185) 10-1 pg 27.27 (0.136) 0.01 N/A 50 28.22 (0.209) 10-2 pg 30.29 (0.136) 0 N/A 10 30.99 (0.284) 10-3 pg 34.75 (0.944) NTC N/A 5 32.43 (0.516) 10-4 pg 36.62 (0.584) 1 33.13 (0.473) 10-5 pg N/A 0.5 34.68 (0.189) 0 N/A 0.1 36.83 (0.589) NTC N/A 0.05 N/A 0.01 N/A 0 N/A NTC N/A
Table 1. Quantitative real-time PCR assay for detection of Thelohanellus kitauei. Mean Cq and SD values were obtained from triplicate runs

NTC, No template control; N/A, No signal was detected within 40 cycles.