脱氮硫杆菌特异引物/探针的设计和评价

自脱氮硫杆菌(Thiobacillus denitrificans)16S rRNA基因V3可变区中发现一条27 bp的特异序列, 以该序列为反向引物, 对高效同步脱硫反硝化系统污泥DNA进行了温度梯度PCR扩增和基因文库构建, 结果证实了该引物的高度专一性。应用该探针在去离子甲酰胺和NaCl的浓度分别为35%和100 mmol/L, 杂交/洗脱温度为48°C条件下对污泥样品杂交得到较好的阳性结果, 软件分析表明脱氮硫杆菌在污泥中约占15%。脱氮硫杆菌专一性引物/探针的提出, 将为不同生态环境中该种微生物的时空分布、结构动态以及实时定量等研究提供分子生物学工具。

Design and Assessment of Species-specific Primer and Probe for Thiobacillus denitrificans

A species-specific oligonucleotide sequence was screened from the third variable domain of 16S rRNA gene of Thiobacillus denitrificans. It confirmed that the sequence had the high speciality by amplifying the activated sludge DNA in high simultaneous biological removal of sulfate and nitrate process and further determining the sequences of 16S rRNA gene clone library. The Cy3-labeled species-specific probe hybridized well with activated sludge and obtained good picture under the conditions of 48°C, 35% formamide deionized, and 100 mmol/L NaCl. ImageJ software demonstrated that T. denitrificans counted for 15% of bacteria in the sludge. The proposal of species-specific primer and probe for T. denitrificans would play an important role in disclosing its temporal and spatial distribution, structure dynamics and realtime quantity.