Development of nicking endonuclease-based technologies for the detec-tion of biomolecules using MALDI-TOF analysis

Abstract

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used for the analysis of nucleic acids, peptides, and proteins with precise and rapid measurement of their mass. MALDI-TOF MS was provided the capability of measuring both low and high molecular mass compound and the benefit to analyze com-plex mixtures. The purpose of this study is development of the diagnosis of genitourinary in-fections and antibiotic resistance marker for the medical treatment of sexually transmitted diseases (STD) by nucleic acid fragment utilizing MALDI-TOF with simplicity and rapidity. In this study, we developed detection method of genitourinary infections by nucleic ac-id fragment using nicking endonuclease. We designed PCR primers containing restriction site to promote activity of nicking endonuclease and mass marker to distinguish various genitouri-nary pathogen at the end of 5’. In the only presence of genitourinary pathogen, amplification of pathogen gene was accomplished resulting that restriction recognition site to accelerate nicking enzyme was incorporated to amplicon. Since nicking endonuclease has a role as cleavage of double-stranded PCR amplicon from genitourinary pathogen, diverse nucleic acid fragments which stand for genitourinary pathogen were generated after amplification of pathogen gene and endonuclease reaction. Following isolation of mass fragment from reac-tion solution, we could diagnose infection through MALDI-TOF measurement. Since STD frequently strike by multiple infections of genitourinary pathogens, it is of importance multiple diagnose of 14 pathogens simultaneously above all. In this study, we have been also performed multiple detection of genitourinary infections. 14 genitourinary pathogens were divided into 3 groups, and we performed multiplex PCR with 3 tubes. As a result, we could detect all selected pathogens.