基于锁核酸的探针在荧光原位杂交中的应用。

Application of locked nucleic acid-based probes in fluorescence in situ hybridization.

机构信息

LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal.

i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.

出版信息

Appl Microbiol Biotechnol. 2016 Jul;100(13):5897-906. doi: 10.1007/s00253-016-7429-4. Epub 2016 Mar 12.

DOI:10.1007/s00253-016-7429-4

PMID:26969040

Abstract

Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2'-O-methyl (2'-OMe) RNA modifications have on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type of nucleic acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2'-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall, these results have significant implications for the design and applications of LNA probes for the detection of microorganisms.

摘要

荧光原位杂交（FISH）采用核酸类似物作为探针，在微生物学领域作为一种新兴的分子工具，用于检测和可视化微生物。然而，锁核酸（LNA）和 2'-O-甲基（2'-OMe）RNA 修饰对靶向微生物的探针的影响尚不清楚。在这项研究中，比较了 18 种不同探针的熔解和杂交效率特性，这些探针用于 FISH 检测幽门螺杆菌的 16S rRNA。对于相同的序列和靶标，探针长度和作为 LNA 探针中混合剂使用的核酸类似物的类型强烈影响检测效率。具有 10 到 15 个核苷酸的 LNA 探针显示出最高的效率。此外，LNA 与 2'-OMe RNA 的组合允许探针的荧光强度增加。总的来说，这些结果对设计和应用 LNA 探针检测微生物具有重要意义。