title: A fully sealed plastic chip for multiplex PCR and its application in bacteria identification.
abstract: Multiplex PCR is an effective tool for simultaneous multiple target detection but is limited by the intrinsic interference and competition among primer pairs when it is performed in one reaction tube. Dividing a multiplex PCR into many single PCRs is a simple strategy to overcome this issue. Here, we constructed a plastic, easy-to-use, fully sealed multiplex PCR chip based on reversible centrifugation for the simultaneous detection of 63 target DNA sequences. The structure of the chip is quite simple, which contains sine-shaped infusing channels and a number of reaction chambers connecting to one side of these channels. Primer pairs for multiplex PCR were sequentially preloaded in the different reaction chambers, and the chip was enclosed with PCR-compatible adhesive tape. For usage, the PCR master mix containing a DNA template is pipetted into the infusing channels and centrifuged into the reaction chambers, leaving the infusing channels filled with air to avoid cross-contamination of the different chambers. Then, the chip is sealed and placed on a flat thermal cycler for PCR. Finally, amplification products can be detected in situ using a fluorescence scanner or recovered by reverse centrifugation for further analyses. Therefore, our chip possesses two functions: 1) it can be used for multi-target detection based on end-point in situ fluorescence detection; and 2) it can work as a sample preparation unit for analyses that need multiplex PCR such as hybridization and target sequencing. The performance of this chip was carefully examined and further illustrated in the identification of 8 pathogenic bacterial genomic DNA samples and 13 drug-resistance genes. Due to simplicity of its structure and operation, accuracy and generality, high-throughput capacity, and versatile functions (i.e., for in situ detection and sample preparation), our multiplex PCR chip has great potential in clinical diagnostics and nucleic acid-based point-of-care testing.
authors.affiliation: School of Medicine, Tsinghua University, Beijing 100084, China. wlxing@tsinghua.edu.cn.
authors.forename: Youchun
authors.initials: Y
authors.lastname: Xu
authors.forename: He
authors.initials: H
authors.lastname: Yan
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Kewei
authors.initials: K
authors.lastname: Jiang
authors.forename: Ying
authors.initials: Y
authors.lastname: Lu
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Ren
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Shan
authors.initials: S
authors.lastname: Wang
authors.forename: Wanli
authors.initials: W
authors.lastname: Xing
mesh_terms: D001419:Bacteria / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D060885:Multiplex Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D009693:Nucleic Acid Hybridization; D010969:Plastics
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D010969:Plastics