title: Multiplex detection and genotyping of pathogenic bacteria on paper-based biosensor with a novel universal primer mediated asymmetric PCR.
abstract: Traditionary multiplex asymmetric polymerase chain reaction (PCR) can be applied to detect multiplex target organisms simultaneously, but complex optimizations of primer concentrations and staggered additions of primers are required to achieve equal amplification of multiplex genes. To overcome this shortcoming, we propose a novel method based on multiplex asymmetric PCR and paper-based nucleic acid diagnostics (PBNAD). In the asymmetric PCR, a universal primer was introduced to break the bottlenecks of low sensitivity and self-inhibition among different sets of primers. Amplification using the novel multiplex asymmetric PCR boosted the quantity of single-stranded amplicons, and the amplified products contained the same sequence at the 5' end. Therefore, only one gold nanoparticle-based signal probe was needed for the simultaneous detection of three genes using the PBNAD platform, and the detection signals could be observed with the naked eye. With this highly efficient, novel multiplex asymmetric PCR, as little as 1 pg/μL genomic DNA can be detected. This method can also be applied to genotyping for reliable epidemiological investigations. This proof-of-concept study highlights the potential of the PBNAD platform for cost- and labor-effective applications in the detection of pathogenic bacteria.
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Liu
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.
authors.forename: Hongxing
authors.initials: H
authors.lastname: Liu
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.
authors.forename: Yuhui
authors.initials: Y
authors.lastname: Liao
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.
authors.forename: Jitao
authors.initials: J
authors.lastname: Wei
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China. Electronic address: zhouxm@scnu.edu.cn.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Zhou
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China. Electronic address: xingda@scnu.edu.cn.
authors.forename: Da
authors.initials: D
authors.lastname: Xing
mesh_terms: D015374:Biosensing Techniques; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D005838:Genotype; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification; D010209:Paper; D016133:Polymerase Chain Reaction; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial
keywords: Genotyping; Multiplex detection; Paper-based nucleic acid diagnostics; Point-of-care application; Universal primer mediated asymmetric PCR