NAIL: Nucleic Acid detection using Isotachophoresis and Loop-mediated isothermal amplification

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Point-of-Care Testing

Background:

• Nucleic acid amplification tests (NAATs) are crucial for infectious disease diagnosis but lack point-of-care (POC) utility due to complex instrumentation and sample preparation.
• Existing NAATs require specialized equipment and extensive lab work, limiting their application in resource-constrained or field settings.

Purpose of the Study:

• To develop an integrated diagnostic device, the Nucleic Acid Isotachophoresis LAMP (NAIL), for rapid and simplified nucleic acid amplification at the point of care.
• To demonstrate the NAIL device's capability for extracting and detecting pathogenic bacteria from complex sample matrices.

Main Methods:

• The NAIL device combines isotachophoresis (ITP) for nucleic acid extraction/purification and loop-mediated isothermal amplification (LAMP) for DNA amplification within a single integrated chip.
• Automated fluid actuation is achieved using capillary valves and heated air chambers, minimizing user intervention.
• Amplification results are detected via mobile phone imaging, eliminating the need for specialized fluorescent readers.

Main Results:

• The NAIL device successfully extracted and detected pathogenic E. coli O157:H7 from whole milk samples.
• The diagnostic limit of detection (LoD) for E. coli in milk was determined to be 1000 CFU mL(-1) using Clinical and Laboratory Standards Institute (CLSI) definitions.
• This LoD represents a two-order-of-magnitude improvement over standard tube-LAMP reactions with diluted milk and is comparable to laboratory-based methods.

Conclusions:

• The NAIL device offers a simplified, rapid, and cost-effective solution for nucleic acid-based diagnostics, significantly improving POC utility.
• This integrated ITP-LAMP system has the potential to revolutionize infectious disease diagnosis by making advanced molecular testing more accessible.
• The technology demonstrates a viable pathway for developing portable diagnostic tools for various complex sample types.