新型 SYBR Green I 与碳纳米管结合策略用于灵敏检测鼠伤寒沙门氏菌 DNA

Novel strategy combining SYBR Green I with carbon nanotubes for highly sensitive detection of Salmonella typhimurium DNA.

机构信息

The Co-construction Laboratory of Microbial Molecular Biology of Province and Ministry of Science and Technology, College of Life Science, Hunan Normal University, Changsha, Hunan, People's Republic of China.

出版信息

Enzyme Microb Technol. 2014 Jan 10;54:15-9. doi: 10.1016/j.enzmictec.2013.09.011. Epub 2013 Oct 1.

DOI:10.1016/j.enzmictec.2013.09.011

PMID:24267562

Abstract

A simple, selective, sensitive and label-free fluorescent method for detecting trpS-harboring Salmonella typhimurium was developed in this study. This assay used the non-covalent interaction of single-stranded DNA (ssDNA) probes with SWNTs, since SWNTs can quench fluorescence. Fluorescence recovery (78% with 1.8 nM target DNA) was detected in the presence of target DNA as ssDNA probes detached from SWNTs hybridized with target DNA, and the resulting double-stranded DNA (dsDNA) intercalated with SYBR Green I (SG) dyes. The increasing fluorescence intensity reached 4.54-fold. In contrast, mismatched oligonucleotides (1- or 3-nt difference to the target DNA) did not contribute to significant fluorescent recovery, which demonstrated the specificity of the assay. The increasing fluorescence intensity increased 3.15-fold when purified PCR products containing complementary sequences of trpS gene were detected. These results confirmed the ability to use this assay for detecting real samples.

摘要

本研究开发了一种简单、选择性强、灵敏度高且无需标记的荧光法来检测含有 trpS 的鼠伤寒沙门氏菌。该测定法利用了单链 DNA（ssDNA）探针与 SWNTs 之间的非共价相互作用，因为 SWNTs 可以猝灭荧光。在存在靶 DNA 的情况下，检测到荧光恢复（用 1.8 nM 靶 DNA 恢复 78%），这是由于 ssDNA 探针从与靶 DNA 杂交的 SWNTs 上脱离，并与 SYBR Green I（SG）染料形成双链 DNA（dsDNA）。荧光强度增加了 4.54 倍。相比之下，错配的寡核苷酸（与靶 DNA 相差 1 或 3 个核苷酸）不会导致显著的荧光恢复，这证明了该测定法的特异性。当检测含有 trpS 基因互补序列的纯化 PCR 产物时，荧光强度增加了 3.15 倍。这些结果证实了该测定法用于检测实际样品的能力。