Real-time quantitative polymerase chain reaction (QPCR) to identify Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss

Diseases of Aquatic Organisms

Kenneth D. Cain¹  John S. Wood¹  Wade P. Cavender¹  Kenneth Overturf¹  Madison S. Powell¹ 

关键词: Myxobolus cerebralis;    Quantitative polymerase chain reaction;    Histopathology;    Hsp 70;    18S rDNA;   

DOI  :  10.3354/dao060205

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

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 cerebralisand 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 chainreaction (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 werecapable 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 strongrelationship 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 higherhistology 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 indicationof infection severity.

【 授权许可】

Unknown