Nitrocellulose-bound achromopeptidase for point-of-care nucleic acid tests

Area of Science:

• Biotechnology
• Enzymology
• Molecular Diagnostics

Background:

• Achromopeptidase (ACP) is crucial for sample preparation in nucleic acid tests, particularly for Gram-positive bacteria.
• ACP's inhibition of DNA amplification complicates its integration into diagnostic workflows.
• Current methods to inactivate ACP, such as heat, are difficult to implement in point-of-care (POC) devices.

Purpose of the Study:

• To develop a method for integrating ACP into POC devices without compromising DNA amplification.
• To investigate the feasibility of immobilizing ACP on nitrocellulose paper for easier handling and activation.
• To overcome the challenge of ACP-mediated inhibition of DNA amplification in diagnostic assays.

Main Methods:

• Immobilization of achromopeptidase (ACP) onto nitrocellulose paper.
• Utilizing recombinase polymerase amplification (RPA) in conjunction with immobilized ACP.
• Activation of immobilized ACP through simple agitation.

Main Results:

• Immobilized ACP on nitrocellulose paper retained its enzymatic activity.
• Activated immobilized ACP efficiently without leaking into the solution.
• This approach eliminated the need for heat-based deactivation of ACP.
• The method successfully integrated ACP with RPA, overcoming amplification inhibition.

Conclusions:

• Nitrocellulose-bound ACP offers a stable and easily activatable solution for sample preparation in POC nucleic acid tests.
• This innovation removes the barrier of ACP inhibition in DNA amplification, paving the way for improved paper-based POC diagnostic devices.
• The developed method simplifies ACP handling and integration, enhancing the potential for rapid, on-site diagnostics.