title: Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level.
abstract: Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100,000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.
authors: State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering; Xiamen University, Xiamen, Fujian, China 361005.
authors: Zhi
authors: Z
authors: Zhu
authors: Wenhua
authors: W
authors: Zhang
authors: Xuefei
authors: X
authors: Leng
authors: Mingxia
authors: M
authors: Zhang
authors: Zhichao
authors: Z
authors: Guan
authors: Jiangquan
authors: J
authors: Lu
authors: Chaoyong James
authors: CJ
authors: Yang
mesh_terms: D048168:Escherichia coli K12 / Q000235:genetics*; D019453:Escherichia coli O157 / Q000235:genetics*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000737:chemistry*
chemical_list: D012685:Sepharose