Functional nucleic acid circuitry as a universal detector for pathogens

Abstract

While the pathogen nucleic acid diagnostic technology has made tremendous progresses, several challenges, including long development cycles and limited specificity still exist, especially in the context of isothermal amplification techniques. To enhance the detection accuracy, here a functional strand displacement catalytic hairpin assembly circuit, which can perform at high-temperature (HT-CHA), was developed as the downstream of the loop mediated isothermal nucleic acid amplification (LAMP). The addition of HT-CHA not only ensures the specificity but also amplifies the detection signal. Taking African swine fever (ASF) gene as the target, the input of HT-CHA was designed according to the ASF gene LAMP amplicons. This LAMP-HTCHA can detect as low as 2 copies/μL targeting genes with high signal-to-noise ratio. Through importing a three-way junction (3WJ) transducer, the HT-CHA well-developed for ASF gene can be directly adapted to detect the LAMP amplicons of other pathogen genes, such as Mycoplasma pneumoniae (MP) gene. The time-consuming and high-risk process to redesign HT-CHA components can be further avoided, making the method even general and useful for a plenty of other targets. Finally, the accurate detection of MP gene in alveolar lavage fluid samples confirmed the high potential of the LAMP and HT-CHA combination in clinical applications, providing a promising strategy to develop point-of-care diagnostics at constant temperatures.

Keywords

• catalytic hairpin assembly
• Loop-mediated isothermal amplification
• Three-way junction
• Universality
• Pathogen detection