Development of Quantitative Real-Time PCR Assays for Postmortem Detection of Mycobacterium spp. Common in Zebrafish (Danio relic) Research Colonies

作者：Meritet Danielle M; Mulrooney Donna M; Kent Michael L; Loehr Christiane V

来源：Journal of the American Association for Laboratory Animal Science, 2017, 56(2): 131-141.

• C-t
• cycle threshold
• FFPE
• formalin-fixed paraffin-embedded
• qPCR
• quantitative real-time polymerase chain reaction
• T-m
• melting temperature

Mycobacterium spp. infections are common in zebrafish kept in research facilities. These comorbidities can substantially modulate the responses of these fish to external and internal stimuli. Therefore, diagnostic tests to detect Mycobacterium spp. infections in zebrafish colonies prove essential. Here, we outline the development of quantitative simplex real-time PCR assays to detect the 3 Mycobacterium species most commonly identified in laboratory zebrafish. The assays targeted the heat shock protein 65 gene of M. marinum, M. chelonae, and M. haemophilum. The assays are both highly specific and sensitive for fresh-frozen samples and highly specific and moderately sensitive for formalin-fixed paraffin-embedded (FFPE) samples. Two sampling techniques for FFPE samples of sagittally sectioned zebrafish were evaluated. Both paraffin cores targeting granulomas containing bacteria and scrolls from the entire fish yielded DNA of equivalent quantity and purity. The diagnostic sensitivity of cores was superior to that of scrolls for M. chelonae and M. haemophilum but not M. marinum. The assays are cost-effective and ideally suited to diagnosing common Mycobacterium spp. infections in zebrafish.

• 出版日期2017-3