Establishment of Multiplex Loop-Mediated Isothermal Amplification for Rapid Detection of Genitourinary Mycoplasma

Background: Genitourinary Mycoplasma (Ureaplasma urealyticum, Mycoplasma hominis, and Mycoplasma genitalium) is a common pathogen among women, which can cause funisitis, spontaneous abortion, and low birth weight. However, current laboratory testing methods for genitourinary mycoplasma normally need complex processes, expensive devices, and qualified staffs, and there are many limits for application. Up to now, the LAMP method is a rapidly developing field because of the significance for clinical application and commercial value. Few studies have reported the use of mLAMP to detect UU, MH, and MG. In this study, a multiplex loop-mediated isothermal amplification system was developed for rapid detection of UU, MH, and MG, concurrently. Methods: Three sets of multiplex LAMP primers were designed to specifically target urease of UU, 16S rRNA of MH, and mgpa of MG. The ratio of primer concentration was optimized. The specificity and sensitivity of multiplex LAMP were explored. Twenty-nine clinical samples were successfully used with mLAMP. Results: In this study, the primer concentration in the mLAMP system was set to 1.3 pmol/L which could maintain reaction efficiency and avoid non-specific reaction. Multiplex LAMP can test UU, MG, and MH simultaneously with high specificity. Meanwhile, the sensitivity of multiplex LAMP was found to be 100 pg for UU, 100 pg for MH and 1 ng for MG, which was much higher than that of conventional PCR Furthermore, among the 29 clinical samples, there were two positive samples determined by mLAMP, which was consistent with the PCR and sequencing results. Conclusions: The multiplex LAMP assay can potentially facilitate simultaneous detection of three kinds of mycoplasma in a large number of samples in clinic, which could be used as a primary screening method and as a supplementary method for classical methods.