Establishment and Application of a Loop-Mediated Isothermal Amplification Method for Simple, Specific, Sensitive, and Rapid Detection of Toxoplasma gondii

抄録

Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high simply, specificity, sensitivity and rapidity. In this study, A LAMP assay with six primers targeting a highly conserved region of the GRA1 gene has been developed to diagnose Toxoplasma gondii. The reaction time of the LAMP assay was shortened to 30 min after optimizing the reaction system. The LAMP assay was found to be highly specific and stable. The detection limit of the LAMP assay was 10 copies, the same as that of the conventional PCR. We used the LAMP assay to develop a real-time fluorogenic protocol to quantitate T. gondii DNA, and generated a log-linear regression plot by plotting the time-to-threshold values against genomic equivalent copies. Furthermore, the LAMP assay was applied to detect T. gondi DNA in 423 blood samples and 380 lymph nodes samples from 10 pig farms. Positive results were 7.8% and 8.2%, anf the result shows LAMP method is some more sensitive than conventional PCR (6.1% and 7.6%). Positive samples obtained from 6 pig farms. The LAMP assay established in this study resulted in simple, specific, sensitive and rapid detection of T. gondii DNA, and is expected to play an important role in the clinical detection.