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DAO 60:205-213 (2004)
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Abstract
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Real-time quantitative polymerase chain reaction (QPCR) to identify Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss
Wade P. Cavender1, John S. Wood2, Madison S. Powell3, Kenneth Overturf3, Kenneth D. Cain1,*
1Department of Fish and Wildlife Resources and the Aquaculture Research Institute, University of Idaho, 6th and Line Street, Moscow, Idaho 83844-1136, USA
2Pisces Molecular LCC, 2200 Central Avenue, Suite F, Boulder, Colorado
80301, USA
3Hagerman Fish Culture Experiment Station, University of Idaho, 3059-F National Fish Hatchery Road, Hagerman, Idaho 83616, USA
*Corresponding author. Email: kcain@uidaho.edu
ABSTRACT: This study describes the development of a TaqMan real-time quantitative polymerase chain reaction (QPCR) technique using the heat-shock protein 70 (Hsp 70) and 18S ribosomal DNA (18S rDNA) sequences to identify Myxobolus cerebralis
and attempt to quantify infection severity within rainbow trout fry Oncorhynchus mykiss. Rainbow trout for this study were exposed to M. cerebralis under natural river conditions and examined for infection by histology, polymerase chain
reaction (PCR) and QPCR analysis at 900 Celsius temperature units (CTUs) following exposure. Detection sensitivity by QPCR was shown to be equal to traditional PCR but greater than histopathology. Primer/probe combinations developed for this study were
capable of specifically detecting M. cerebralis DNA in infected fish tissue and single triactinomyxon (TAM) spores with a sensitivity of 12.5 and 6.3 pg µl–1 of DNA for the Hsp 70 and 18S rDNA sequences, respectively. A strong
relationship between QPCR and infection severity was found for the Hsp 70 probe when parasite copy number and histology scores of 0–4 were compared (R2 = 0.96, p = 0.003). However, a reduction in copy number was observed at higher
histology scores for the 18S probe (scores of 4 and 5) and the Hsp 70 probe (score of 5). The results of this study demonstrate that QPCR analysis is an effective tool for detecting M. cerebralis in fish tissue and may provide a relative indication
of infection severity.
KEY WORDS: Myxobolus cerebralis · Quantitative polymerase chain reaction · Histopathology · Hsp 70 · 18S rDNA
Full text in pdf format
Published in DAO Vol.
60, No. 3
(2004) on September 8
Print ISSN: 0177-5103; Online ISSN: 1616-1580.
Copyright © Inter-Research, Oldendorf/Luhe, 2004
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