Development Of Detecting The Infection Of Toxoplasma Gondii By LAMP

Toxoplasma gondii, as an obligate intracellular parasite, it can cause epidemical parasitic disease. As toxoplasmosis could infect humans and all other warm-blooded animals, it not only threatens humans'health, but also causes great economic losses on storkbreeding and was considered to be one of the most widespread parasites in the world. Among the three transmission ways of toxoplasmosis, fecal-oral is the most common way.The traditional diagnostic methods of toxoplasmosis are mainly based on etiology and immunology. With the development of molecular biology techniques, the diagnosis has improved a lot. According to the nucleoside triphosphate hydrolase gene sequence, which comes from the GJS strain of Toxoplasma gondii, six specific primers were designed to develop the method of loop-mediated isothermal amplification (LAMP), and the specificity and sensitivity was then detected. Results showed that, the nucleoside triphosphate hydrolase gene could be amplified by LAMP in just 30 minutes, and the sensitive threshold of recombinant plasmid DNA detection was 20 copies, which was 10 times of that by PCR method. There was no cross-reaction with Neospora caninum, Chlamydophilia psittaci, Eimeria tenella, Theileria luwenshuni, Babesia sp., Babesia bovis, Anaplasma marginale, Mycoplasma mucoidessushsp. Capri, B. burgdorferi sensu strict, Haemophilus parasuis and Streptococcus suis. The reproducibility was also confirmed well.After inoculation to the exprienmental anminals of pigs and rabbits with a certain dose of T. gondii, the blood and tissus samples were detected by LAMP. In the blood samples of pigs, T. gondii could be detected just in 2 days by LAMP, while it was 4 days by PCR method. In the early diagnosis experiment, T. gondii could be detected in the spleen, lung by LAMP at the first day after infection with T. gondii, while it was 2 days by PCR method. Heart, kidney and muscle were detected with T. gondii by LAMP in 2 days by LAMP, which was 1 day earlier than PCR. Liver and lymph were all detected with T. gondii in 2 days after infection using the two methods.The blood samples from 185 sheep, 17 schizophrenia, and tissue samples from 43 chicken in Gansu province in China were detected by the established LAMP method. The results showed that the infection rates with T. gondii were 20.0%, 52.9%, 18.6%, respectively.In this study,the LAMP with specificity and sensibility was established to diagnosis toxoplasmasis. And the early diagnosis of pigs and rabbits infected with T. gondii and investigated the epidemiology of toxoplasmasis were also using this method, which lays the foundation to support the development of rapid diagnostic kits of toxoplasmosis.