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KMID : 0545120180280111823
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Journal of Microbiology and Biotechnology
2018 Volume.28 No. 11 p.1823 ~ p.1833
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Design, Optimization and Verification of 16S rRNA Oligonucleotide Probes of Fluorescence in-situ Hybridization for Targeting Clostridium spp. and Clostridium kluyveri
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Hu Lintao
Huang Jun
Li Hui
Jin Yao
Wu Chongde
Zhou Rongqing
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Abstract
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Fluorescence in-situ hybridization (FISH) is a common and popular method to investigate microbial communities in natural and engineered environments. In this study, two specific 16S rRNA-targeted oligonucleotide probes CLZ and KCLZ were designed and verified to quantify the genus Clostridium and the species Clostridium kluyveri. The optimal concentration of hybridization buffer solution for both probes CLZ and KCLZ was 30% (w/v). The specificity of the designed probes was high by using pellets from the pure reference strains. Feasibility was tested using the samples of Luzhou-flavor liquor manufacturing cellar. The effectiveness of detecting target cells appears to vary widely in different environments. In pit mud, the detection effectiveness of the target cell by probe CLZ and KCLZ was 49.11% and 32.14%, respectively. The quantitative analysis by FISH technique of microbes in pit mud and fermented grains showed consistency with the results detected by qPCR and PCR-DGGE techniques, which showed that the probes CLZ and KCLZ were suitable to analyze the biomass of Clostridium spp. and C. kluyveri during liquor fermentation. Therefore, this study provides a method for quantitative analysis of Clostridium spp. and C. kluyveri and monitoring their community dynamics in microecosystems.
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KEYWORD
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Fluorescence in-situ hybridization, 16S rRNA, specific oligonucleotide probe, clostridium, clostridium kluyveri
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