A single-tube sample preparation method based on a dual-electrostatic interaction strategy for molecular diagnosis of gram-negative bacteria

Abstract

A single-tube method based on a dual-electrostatic interaction (EI) strategy for bacteria capture and DNA extraction was designed to enable the highly sensitive detection of nucleic acids. Specially designed magnetic nanoparticles were developed to meet the opposing requirements of a single-tube method, which exist between the strong EI required for efficient bacteria capture and the weak EI required for DNA extraction with minimal DNA adsorption. A dual-EI strategy for the single-tube (DESIGN) method was thus developed to integrate bacteria enrichment, bacteria cell lysis, and DNA recovery in a single tube, thereby minimizing precious sample loss and reducing handling time. Subsequently, we evaluated the performance with a variety of concentrations from 5 to 100 colony-forming units (CFU)/10 mL human urine and milk samples. The DESIGN method achieved the simple and sensitive detection of Salmonella enterica serovar Typhimurium in 10 mL of human urine and milk samples up to 5 CFU by quantitative PCR. Furthermore, the DESIGN method detected Brucella ovis and Escherichia coli from 10 mL of human urine with a detection limit up to 5 CFU/10 mL.

Graphical abstract

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Abbreviations

EI:

Electrostatic interaction

MNP:

Magnetic nanoparticle

DESIGN method:

Dual-electrostatic interaction strategy based on a single-tube method

S. Typhimurium:

Salmonella enterica serotype Typhimurium

B. ovis :

Brucella ovis

E. coli :

Escherichia coli

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Acknowledgments

The authors gratefully acknowledge Center for Bio-Medical Engineering Core Facility at Dankook University for providing critical reagents and equipment.

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2019R1I1A3A01063051) and a research fund from Chungnam National University.

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Correspondence to Kyudong Han or Tae Yoon Lee.

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Chen, F., Kim, S., Na, JH. et al. A single-tube sample preparation method based on a dual-electrostatic interaction strategy for molecular diagnosis of gram-negative bacteria. Microchim Acta 187, 558 (2020). https://doi.org/10.1007/s00604-020-04536-9

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Keywords

  • Electrostatic interaction
  • Magnetic nanoparticle
  • Pathogenic bacteria enrichment
  • Pathogenic bacteria detection