The Application of Reverse Transcription Loop-mediated Isothermal Amplification to Detect Cell-Free Seminal mRNA

Cell-free seminal mRNA (cfs-mRNA) has been utilized in the diagnosis of diseases affecting the male reproductive system. However, the existing detection methods are time-consuming and labor-intensive. Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method with high efficiency and accuracy; however, it has not been applied to cell-free RNA in human body fluids. Herein, we designed gene-specific primers for TGM4 and DDX4 and established a reverse transcription-LAMP (RT-LAMP) system for detecting their cfs-mRNA. The sensitivity of this system was investigated using RNA standards. The optimal conditions were 65 ℃ for 30 to 45 min for TGM4 and 65 ℃ for 45 to 60 min for DDX4. When incubated at 65 ℃ for 45 min, the limit of detection (LOD) for DDX4 mRNA was 124 copies, which increased to 37 copies when the reaction time was extended to 90 min. We also validated the RT-LAMP reaction using semen plasma samples from 20 azoospermic patients and identified 3 DDX4 cfs-mRNA negative patients among 72 azoospermic patients in total. In conclusion, this study establishes a novel RT-LAMP method for rapid and sensitive detection of TGM4 and DDX4 cfs-mRNA in seminal plasma. RT-LAMP is a rapid, sensitive and specific method for cfs-mRNA detection, which has the potential to be used in the diagnosis of male reproductive system diseases, RNA detection in other body fluids, and commercial promotion. The validated technique offers significant potential for efficient diagnosis of male reproductive disorders and broader RNA detection applications.