Direct fluorescence in situ hybridization (FISH) in Escherichia coli with a target-specific quantum dot-based molecular beacon.
Quantum dots (QDs) are inorganic fluorescent nanocrystals with excellent properties such as tunable emission spectra and photo-bleaching resistance compared with organic dyes, which make them appropriate for applications in molecular beacons. In this work, quantum dot-based molecular beacons (QD-based MBs) were fabricated to specifically detect β-lactamase genes located in pUC18 which were responsible for antibiotic resistance in bacteria Escherichia coli (E. coli) DH5α. QD-based MBs were constructed by conjugating mercaptoacetic acid-quantum dots (MAA-QDs) with black hole quencher 2 (BHQ2) labeled thiol DNA vial metal-thiol bonds. Two types of molecular beacons, double-strands beacons and hairpin beacons, were observed in product characterization by gel electrophoresis. Using QD-based MBs, one-step FISH in tiny bacteria DH5α was realized for the first time. QD-based MBs retained their bioactivity when hybridizing with complementary target DNA, which showed excellent advantages of eliminating background noise caused by adsorption of non-specific bioprobes and achieving clearer focus of genes in plasmids pUC18, and capability of bacterial cell penetration and signal specificity in one-step in situ hybridization.
Wu
Sheng-Mei
SM
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, Wuhan University, Wuhan 430072, PR China.
Tian
Zhi-Quan
ZQ
Zhang
Zhi-Ling
ZL
Huang
Bi-Hai
BH
Jiang
Peng
P
Xie
Zhi-Xiong
ZX
Pang
Dai-Wen
DW
D015342:DNA Probes / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques; D045663:Quantum Dots; D001618:beta-Lactamases / Q000235:genetics*
D015342:DNA Probes; D001618:beta-Lactamases