Bioluminescence (BL) imaging, which utilizes light emitted through the enzymatic reaction of luciferase oxidizing its substrate luciferin, enables sensitive and noninvasive monitoring of life phenomena. Herein, we developed a series of caged furimazine (FMZ) derivatives by introducing a protective group at the C-3 position and a hydroxy group at the C-6 phenyl ring to realize long-term live-cell BL imaging based on the NanoLuc (NLuc)/ NanoKAZ (NKAZ)−FMZ system. The membrane permeability and cytotoxicity of the substrates were evaluated and related to their hydrophobicity. Among the series, the derivative with the bulkiest protective group (adamantanecarbonyl group) and a hydroxy substituent (named Ad-FMZ-OH) showed significantly prolonged and constant BL signal in cells expressing NLuc compared to the native FMZ substrate. This derivative enabled continuous BL imaging at the single-cell level for 24 h. Furthermore, we applied Ad-FMZ-OH to BL imaging of myocyte fusion and succeeded in the consecutive and sensitive monitoring at a single-cell level over a day. In summary, NLuc/NKAZ-caged FMZ derivatives have the potential to be applied to live-cell BL imaging of various life phenomena that require long-term observation.
A quantitative PCR (qPCR) assay using SYBR Green I was developed based on the published sequence of the gtxA gene from Gallibacterium anatis. This method produced reliable specificity, sensitivity, and repeatability. The detection rate of Gallibacterium in 181 clinical samples was 36.5% (66/181) by qPCR, which was superior to the detection rate of Gallibacterium-specific PCR (0/181) and an isolation and identification assay (18.2% or 33/181). No association was found between the prevalence of Gallibacterium and the age of the chickens. Gallibacterium infection was detected in one 4-day-old chicken, showing that infection can occur much earlier than the previously stated fourth week of life. Tissue sample analysis showed that Gallibacterium is mainly located in the trachea and ovaries, based on results from three groups of chicken with different health statuses. Furthermore, a titer analysis suggested that Gallibacterium loads in different organs may correlate with different clinical manifestations of disease. Thus, the qPCR assay developed in the present study is useful for identification and quantitative analysis of gtxA-containing Gallibacterium in various tissue samples from birds and for the assessment of the pathogenic mechanisms of Gallibacterium.
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