Bioluminescence Assay for Highly Sensitive Detection of Pathogen DNA Directly from PCR Products via Cell-Free Synthesis of a Fragment of NanoLuc Luciferase

Biosensing of nucleic acids by reporter proteins has the potential to outperform traditional fluorophore-based nucleic acid detection systems. However, its complexity and low sensitivity limit utility, particularly in clinical settings. Here, we devised a method that couples PCR amplification to cell-free synthesis of a HiBiT peptide, which is fragment of NanoLuc luciferase. This is achieved by tailing PCR primers with sequences required for in vitro transcription and translation of HiBiT. This method, named HiBiT-PCR, allowed us to generate NanoLuc luciferase directly from the PCR product, with a detection limit for Mycobacterium tuberculosis DNA as low as 1 copy/μL. Furthermore, we validated HiBiT-PCR using clinical samples. The results show that HiBiT-PCR is a highly sensitive and streamlined method for detecting pathogen DNA.