Detection of Toxoplasma gondii based on sequence r529 and sag1 gene probe

Abstract

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects all warm- blooded animals, including humans. It is the pathogenic agent of toxoplasmosis which is one of the main causes of infectious reproductive failure in small ruminants in the world. It causes fetal resorption, abortion, stillbirth and neonatal mortalities resulting in great economic losses. Diagnostic tool based on nucleic acid is an attractive alternative for parasite detection. Several target genes of T. gondii have been cloned and expressed. Two kinds of potential target sequence for detection are sequence repetitive 529 bp (R529) and Surface antigen1 (SAG1) gene. SAG1 is the most immunodominant and stage-specific antigen of tachyzoite and is highly conserved in most isolated T. gondii strains examined. SAG1 has been proven to be a good candidate for diagnosis and vaccine development. R529 is a high copy number fragment and conserved within genom of T. gondii during evolution. The nucleotide sequence of R529 and SAG1 gene has been established. The purpose of this study is to developed probe from R529 and SAG1 gene that have high specificity and sensitivity. R529 and SAG1 was isolated from genome of T. gondii and Amplified by PCR using sense and antisense to derived 237 bp (probe-TR) and 213 bp (probe-TS), respectively. The sequence was labeled with anti-digoxigenin (non radioactive labeled) using PCR Dig Labeling Mix. The dilution of probe-TR can be detect until the quantities of 2,9 pg whereas probe TS until 56.7 pg/µl.