Isothermal DNA amplification using double DNA primer

Nucleic acid amplification techniques have revolutionized diagnostics of human diseases. However, the gold standard, polymerase chain reaction (PCR)-based tests, require specialized laboratories and highly qualified personnel, which delays delivering test results to patients and health care providers. Existing isothermal amplification techniques are compatible with point-of-care settings but suffer from low specificity, require complex primer design and reaction optimization, and have limited multiplexing capabilities. Here, we propose an alternative isothermal amplification technique to solve the limitation of existing methods for isothermal amplification. The technique uses a pair of double-stranded primers that can be elongated by DNA polymerases with strand displacement activity currently used in isothermal techniques (e.g. in LAMP). Each of the two fragments of the double-stranded primer can start the newly synthesized sDNA strands, which would produce high concentrations of the amplified DNA. The reaction will produce a double-stranded DNA amplicon of a defined size, similarly to the products of PCR. The dsDNA amplicon can be detected with sequence-specific probes in a multiplex format, similar to qPCR. It is expected that the new method will contribute to the development of accurate, rapid and affordable point-of-care and home tests for diagnosis of human diseases. The technology can impact other fields including fundamental research, environmental monitoring, food monitoring and forensic applications, among others. Project Number: 1R03AI193602-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Dmitry Kolpashchikov | Institution: UNIVERSITY OF CENTRAL FLORIDA, ORLANDO, FL | Award Amount: $151,496 | Activity Code: R03 | Study Section: Enabling Bioanalytical and Imaging Technologies Study Section[EBIT] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R03AI19360201