Evaluation of the real-time fluorescence loop-mediated isothermal amplification assay for the detection of Ureaplasma urealyticum

Abstract

Abstract Ureaplasma urealyticum (UU) is commonly present in human reproductive tract, which frequently leads to genital tract infection. Hence, there is an urgent need to develop a rapid detection method for UU. In our study, a real-time fluorescence loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of UU. Two primers were specifically designed based on the highly conserved regions of ureaseB genes. The reaction was carried out for 60 min in a constant temperature system using Bst DNA polymerase, and the process was monitored by real-time fluorescence signal, while polymerase chain reaction (PCR) was performed simultaneously. In real-time fluorescence LAMP reaction system, positive result was only obtained for UU among 9 bacterial strains, with detection sensitivity of 42 pg/μL (4.2 × 105 CFU/mL), and all 16 clinical samples of UU could be detected. In conclusion, real-time fluorescence LAMP is a simple, sensitive, specific and effective method compared with conventional PCR, which shows great promise in the rapid detection of UU.

Keywords

• Ureaplasma urealyticum
• Loop-mediated isothermal amplification
• Real-time