Rapid and Visual Detection of Toxoplasma gondii in Blood Samples from Pet Cats and Dogs by Loop-Mediated Isothermal Amplification.

Abstract

Background: Toxoplasma gondii is an obligate intracellular parasite that invades nearly all nucleated cells of a broad spectrum of vertebrate hosts, and which may cause serious disease in immunocompromised patients, as well as in the immunologically incompetent fetus. This study aimed to establish a loop-mediated isothermal amplification (LAMP) technique to rapidly detect T. gondii in the blood infection by targeting the 529 bp repeat element of T. gondii. Materials and Methods: A turbidity monitoring system, together with visual reagent, was used to test the amplification result of the LAMP assay. In addition, the specificity and sensitivity of the LAMP assay were measured. Results: The results suggest that the successfully established LAMP assay profile can detect the DNA of T. gondii at 67°C within 40 min. The limit of detection of the LAMP assay was 101 copies/μL. No cross reaction occurred with Plasmodium vivax, Toxocara cati, Clonorchis sinensi, Spirometra mansoni or Cryptosporidium parvum. We validated the developed LAMP assay by detecting T. gondii in DNA extracted from 353 blood samples collected from domestic cats and dogs. The percentages of positive results in detecting these blood samples by LAMP and conventional PCR were 5.38% and 2.83%, respectively. Conclusions: Our findings show that the developed LAMP assay offers higher analytical sensitivity than conventional PCR and good analytical specificity, minimizes aerosol contamination, and can be applied to on-site rapid detection of T. gondii.