Multiplex detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.
A simple, highly sensitive, and specific assay was developed for the homogeneous and multiplex detection of microRNAs (miRNAs) by combining molecular beacon (MB) probes and T7 exonuclease-assisted cyclic amplification. An MB probe with five base pairs in the stem region without special modification can effectively prevent the digestion by T7 exonuclease. Only in the presence of target miRNA is the MB probe hybridized with the target miRNA, and then digested by T7 exonuclease in the 5' to 3' direction. At the same time, the target miRNA is released and subsequently initiates the nuclease-assisted cyclic digestion process, generating enhanced fluorescence signal significantly. The results show that the combination of T7 exonuclease-assisted cyclic amplification reaction and MB probe possesses higher sensitivity for miRNA detection. Moreover, multiplex detection of miRNAs was successfully achieved by designing two MB probes labeled with FAM and Cy3, respectively. As a result, the method opens a new pathway for the sensitive and multiplex detection of miRNAs as well as clinical diagnosis. Graphical Abstract A simple, highly sensitive, and specific assay was developed for the detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, Hebei, 071002, China.
Yacui
Y
Liu
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, Hebei, 071002, China. hdzjy2005@163.com.
Jiangyan
J
Zhang
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, Hebei, 071002, China.
Jingxiao
J
Tian
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, Hebei, 071002, China.
Xiaofei
X
Fan
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, Hebei, 071002, China.
Hao
H
Geng
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, Hebei, 071002, China. yqcheng@hbu.edu.cn.
Yongqiang
Y
Cheng
D005090:Exodeoxyribonucleases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D015335:Molecular Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods
D035683:MicroRNAs; D015335:Molecular Probes; D005090:Exodeoxyribonucleases; C046264:gene b exonuclease
Molecular beacon; Multiplex microRNA detection; Nuclease-assisted cyclic amplification; T7 exonuclease