title: Real-time monitoring of mycobacterium genomic DNA with target-primed rolling circle amplification by a Au nanoparticle-embedded SPR biosensor.
abstract: In this study, we developed a surface plasmon resonance (SPR) DNA biosensor array based on target-primed rolling circle amplification (RCA) for isothermal and rapid detection of two pathogenic mycobacteria, Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC).The species-specific padlock probe (PLP) was designed to target the sequence in 16S-23S rRNA gene internal transcribed spacer (ITS). After ligation, the circularized PLP could be primed by the target sequence to initial RCA. The RCA performed simultaneously with the cleavage reaction to produce small fragments of single strand DNA which immediately hybridized with the probe immobilized on the sensor chip without denaturation. This process caused SPR angle changes on the chip surface, which made the detection for analysis from the solution achievable, and dynamic real-time RCA monitoring of mycobacterium possible. Besides, Au nanoparticles (AuNPs) were directly assembled onto the surface of the sensor chip via hexanedithiol (HDT) for the enhancement of sensitivity as a label-free detection system. Experimental results show that the signal enhancement by the target-primed RCA together with AuNPs-embedded surface caused at least10-fold increased sensitivity as compared with conventional RCA on bare SPR chip method. Within 40min amplification duration as low as 20amol of synthetic targets and 10(4)CFUmL(-1) of genomic DNA from clinical samples can be detected. The proposed method not only provides a simple design idea for liquid-phase amplification monitoring, but also apply it in clinical pathogen detection, which holds great promise in ultrasensitive bioassay in the future.
authors.affiliation: Department of Clinical Laboratory Science, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xiang
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Huang
authors.affiliation: Department of Clinical Laboratory Science, Southwest Hospital, Third Military Medical University, Chongqing 400038, China. Electronic address: zhengalpha@yahoo.com.
authors.forename: Junsong
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, China. Electronic address: weilingfu@yahoo.com.
authors.forename: Weiling
authors.initials: W
authors.lastname: Fu
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D003199:Computer Systems; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics*; D006046:Gold; D006801:Humans; D053768:Metal Nanoparticles; D009161:Mycobacterium / Q000235:genetics* / Q000302:isolation & purification*; D015269:Mycobacterium avium Complex / Q000235:genetics / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D015345:Oligonucleotide Probes / Q000235:genetics; D013045:Species Specificity; D020349:Surface Plasmon Resonance / Q000379:methods
chemical_list: D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D006046:Gold
keywords: Au nanoparticle-embedded; Mycobacterium; Real-time monitoring; Surface plasmon resonance; Target-primed rolling circle amplification