Isothermal Nucleic Acid Amplification Combined with Gold Nanoparticles Assisted Electrochemical Impedance for the Sensitive and Efficient Pdcov Detection

The emergence of porcine delta coronavirus (PDCoV) has caused huge economic losses in the global pig industry. How to realize the sensitive and efficient detection for it is a difficult problem that need to be resolved. In this work, loop-mediated isothermal amplification (LAMP)-electrochemical impedance spectroscopy (EIS) detection platform for PDCoV based on nucleic acid level was constructed by combining the advantages of efficient amplification for LAMP and sensitive detection for EIS. Referring to a 159 bp fragment of PDCoV N gene (Genbank:KY078891, 641 bp-799 bp), primers (SH-FIP、BIP、F3、B3) were designed to screened and sulfhydryl groups were activated, and then loop-mediated isothermal amplification was carried out. Subsequently, gold nanoparticles were loaded on indium tin oxide glass by electrodeposition technology, and the amplified products were connected to the electrode surface through the formation of Au-S bonds. According to the difference of charge transfer resistance after double-stranded DNA was connected on the electrode surface, the detection platform can achieve valid detection of PDCoV in the concentration range of 102-107 copies/μL, the limit of detection is 28 copies/μL, and can be used for practical analysis of pig small intestine samples.