Novel enzymes enable point-of-care isothermal amplification of DNA/RNA pathogens

Overview

AmpliFast is an isothermal nucleic acid amplification testing system designed for rapid, point-of-care detection of DNA and RNA pathogens. The technology uses engineered recombinase enzymes, single-stranded DNA-binding proteins, strand-displacing polymerase, and target-specific primers to exponentially amplify nucleic acids at a single ambient temperature of 37–42°C, without the need for thermal cycling. Results are read visually on a lateral flow strip.

The current AmpliFast system delivers DNA/RNA detection in approximately 30 minutes from sample collection to results. The next-generation AmpliFast 2.0 will incorporate lyophilized mixtures of enzymes, primers, and buffers to eliminate sample processing and reduce total time to results to approximately 20 minutes. The lyophilized format will also extend compatibility to whole blood and urine specimens, broadening the range of clinical and field settings where the test can be deployed.

Technical specifications

Core technology:

• Recombinase polymerase amplification (RPA) performed at a single temperature between 37°C and 42°C
• Novel engineered recombinase and single-stranded DNA-binding proteins (SSBP) covered by a provisional patent
• Strand-displacing DNA polymerase for exponential amplification
• Reverse transcriptase included for RNA targets such as SARS-CoV-2
• Target-specific forward and reverse primers optimized for low-temperature amplification within 10 minutes
• Lateral flow strip readout for visual interpretation without instrumentation

AmpliFast 2.0 enhancements:

• Lyophilized enzyme and primer chemistry that removes the need for sample processing
• Compatibility with whole blood and urine in addition to nasal swab specimens
• Detection targets: gonorrhea, chlamydia, syphilis, and herpes simplex viruses
• Total time to results reduced from 30 minutes to approximately 20 minutes

Performance characteristics:

• Sensitivity and specificity comparable to or exceeding laboratory nucleic acid tests
• Published demonstration of superior sensitivity compared to antigen-based lateral flow tests for SARS-CoV-2 detection

Technology readiness level

AmpliFast has been validated for SARS-CoV-2 detection from nasal swab specimens, with published results demonstrating higher sensitivity than antigen-based lateral flow testing. The provisional patent on the novel recombinase and SSBP enzymes provides early intellectual property protection.

Future validation efforts will proceed in three aims over a one-year period. First, lyophilized chemistries will be optimized using pUC19 DNA as a positive control. Second, primer pairs specific to gonorrhea, chlamydia, syphilis, and herpes simplex viruses will be identified and confirmed compatible with the lyophilized format, capable of amplification at low temperatures within 10 minutes, and readable on lateral flow strips without non-specific signal. Third, the tests will be validated using retrospective, de-identified whole blood and urine specimens. Upon completion, AmpliFast 2.0 is expected to be positioned for pilot deployment and further clinical validation toward point-of-care diagnostic use.