Effect of 3D branched crystalline carbon nitride on nucleic acid amplification techniques in food samples

Abstract

The accurate and rapid detection of foodborne pathogens based on nucleic acid amplification techniques (NAATs) is urgently needed for public health. However, the low specificity and poor sensitivity, resulting from the formation of hairpin structures, false positive, primer dimers, mismatched hybridization, reaction buffer content, and low thermal conductivity severely restrict their real-world applications. Herein, the 3D branched crystalline carbon nitride with 1D nanoneedles (3DBC-C₃N₄)-based four kinds of NAATs including polymerase chain reaction (PCR), accelerated strand exchange amplification (ASEA), loop-mediated isothermal amplification (LAMP) and strand exchange amplification (SEA) were investigated for rapid, specific, and quantitative detection of pathogens, due to its high adsorption affinity for primers and DNA staining dyes, which suppresses the background fluorescence signal, interacts with the polymerase, and enhances thermal conductivity. The 3DBC-C₃N₄ with unique structures possesses abilities such as adsorption, release, restriction, and good thermal conductivity. Furthermore, its nanoconfined environment enhances the enzymatic amplification process. Thus, a faster amplification rate, specific amplification and higher products yields were obtained, allowing quantitative detection of V. parahaemolyticus in food samples down to 10° CFU/g for PCR, 10³ CFU/g for ASEA, 10¹ CFU/g for LAMP and 10³ CFU/g for SEA, which was one or two orders of magnitude lower compared with the classic assay of PCR, SEA, and LAMP. In addition, 3DBC-C₃N₄ enhanced the effect of multiple rounds of PCR amplification. The impacts of 3DBC-C₃N₄ on the efficiency and specificity of various NAATs provide a method for pathogen testing in food safety.