Development And Preliminary Application Of PCR And ELISA Diagnostic Methods Of Toxoplasmosis

Toxoplasma is an obligate intracellular parasite, and it infects majority mammals. It threatens the pregnant animals, and causes grave loss for industry of livestocks. Because the toxoplasmosis lack peculiar clinical symptom, diagnosis is still an serious problem for it. The serum detection method is common used on clinical and recommended by hygiene department of our country recently. However,there are some problem ,such as cross reaction, false positive and negative. Compared with some kits abroad, the kits developed in our country is still unsatisfied. The main problem is low antigen activation and purity. For this reason, we explore the feasibility of developing ELISA kit by using the expression protein of SAG â…  gene in E.coli. At the same time, the PCR method for toxoplasma gondii was also developed and used on clinical.1. Establishment and application of Toxoplasmosis PCR According to the sequence of SAG I published in Genebank, a pair primers wasdesigned for amplifying the conservative sequence of SAG â…  gene, and the length is 270bp.After defining the suitable condition , we establish the PCR method of Toxoplasma gondii. This method can detecte at least the DNA of 0.85 Toxoplasma gondii. Positive results only found in Toxoplasma gondii samples and didn't found in other pathogen. 40 positive of 85 samples from clinical sick swine were detected by PCR. Theses results showed that the PCR method had the specificity and high sensitivity.2. The construction and expression of recombinant plasmids of SAG â…  gene of Toxoplasma gondii and analysis the antigenic ofexpression productAccording to the sequence of SAG â… , we design and synthesize a pair primes .The 978bp fragment of SAG â…  gene was obtained by PCR, its nuclectide sequence was determined by the dideoxy-mediated chain termination method. Then, the DNA fragment of SAG â…  gene was subcloned into prokaryotic expression vector pGEX-KG, the specific fusion protein with GST of molecular weight 56KD was expressed in E.coliBL21 (DE3). Western blotting assay indicated that the polyclonal antibody against Toxoplasma gondii could recognize this protein. This study will give some help on studying epidemiology, vaccine, diagnosis and control of Toxoplasmis.3. Establishment and application of ELISASAG I gene of Toxoplasma gondii was expressed by E.coli BL21(DE3). After the conditions of expression were optimized, the inclusion bodies were purified. The suitable concentration of antigen was determined by square matrix titration, and the specificity, sensitiveness, repetitiveness of this method were determined. 300 serum samples from pig farms were tested by IHA and ELISA at the same time, the coincidence rate is up to 98%,the differencre between the two methods is not remarkable. These Tesults indicate that the ELISAhas high specificity, sensitiveness and good repetitiveness.