Development of an efficient multiplex semi-nested PCR for convenient use in urine samples for diagnosis of toxoplasmosis

Abstract

BACKGROUND: Toxoplasmosis is a protozoan parasitic infection. The cerebrospinal, ocular and congenital forms of the disease are complicated and life threatening. Diagnosis of toxoplasmosis is difficult due to invasive sample requirement, complications in pathogenesis, immunology, and interpretation of test results. Using urine as a non invasive sample source for molecular diagnosis of toxoplasmosis is the main object of this research. Attempts were made to diagnose toxoplasmosis using polymerase chain reaction (PCR) technique on the parasite’s deoxyribonucleic acid (DNA) in urine. METHODS: Parasite: The Toxoplasma gondii (T. gondii) RH strain was obtained from peritoneal exudates of small white laboratory mice inoculated with 5x105 tachyzoites three days before aspiration. Urine samples with defined numbers of tachyzoites per ml were used as laboratory samples. The target was a 529 bp segment (AF146527 gene bank) of the T. gondii genome with 200-300 repeats as target in PCR. Selected primers were designed on its sequence. RESULTS: A multiplex semi-nested PCR technique was developed for obtaining a method for precise diagnosis of toxoplasmosis, time and budget saving and to diminish the DNA cross contamination risk. It was sensitive to detect 2 this tachyzoites DNA per final sample. CONCLUSION: With further development, nested PCR can be a useful and non-invasive method for diagnosis of cerebrospinal, ocular, and congenital toxoplasmosis.