Mona K Hegazy1, Soha I Awad1, Nora E Saleh2, Mamdouh M Hegazy1. 1. Department of Medical Parasitology, Faculty of Medicine, Mansoura University, Mansoura, 35516, Egypt. 2. Department of Medical Parasitology, Faculty of Medicine, Mansoura University, Mansoura, 35516, Egypt. Electronic address: noraesam00@yahoo.com.
Abstract
INTRODUCTION: The cosmopolitan protozoan Toxoplasma gondii is a major parasite of warm-blooded animals including man. Early and accurate diagnosis is a must for proper treatment that prevents life threatening sequels. Loop-mediated isothermal amplification (LAMP) is a novel technique that can amplify DNA with high sensitivity and specificity under isothermal conditions. AIM OF THE STUDY: To validate a LAMP-specific protocol for detection of Toxoplasma DNA using dried blood spots (DBS) from mice experimentally infected with the cystogenic Toxoplasma ME-49 strain. METHODS: In this study, the target DNA fragment was the Toxoplasma 529-bp repeat element that exists in 200-300 copies per T. gondii genome. The sensitivity of both LAMP and conventional PCR techniques was estimated in DBS samples from experimental mice at 1-week and 8-weeks post-infection. RESULTS: Out of 20 blood samples gathered on Whatman filter paper from mice at 1-week post-infection, 18 and 16 were positive by LAMP and conventional PCR, respectively. Neither techniques detected parasite DNA in blood at 8th week of infection. CONCLUSION: Dried blood spots are easy source of material for molecular studies. LAMP assay proved higher sensitivity than the conventional PCR in detecting parasitemia in early infection with the cystogenic Toxoplasma strain.
INTRODUCTION: The cosmopolitan protozoan Toxoplasma gondii is a major parasite of warm-blooded animals including man. Early and accurate diagnosis is a must for proper treatment that prevents life threatening sequels. Loop-mediated isothermal amplification (LAMP) is a novel technique that can amplify DNA with high sensitivity and specificity under isothermal conditions. AIM OF THE STUDY: To validate a LAMP-specific protocol for detection of Toxoplasma DNA using dried blood spots (DBS) from mice experimentally infected with the cystogenic Toxoplasma ME-49 strain. METHODS: In this study, the target DNA fragment was the Toxoplasma 529-bp repeat element that exists in 200-300 copies per T. gondii genome. The sensitivity of both LAMP and conventional PCR techniques was estimated in DBS samples from experimental mice at 1-week and 8-weeks post-infection. RESULTS: Out of 20 blood samples gathered on Whatman filter paper from mice at 1-week post-infection, 18 and 16 were positive by LAMP and conventional PCR, respectively. Neither techniques detected parasite DNA in blood at 8th week of infection. CONCLUSION: Dried blood spots are easy source of material for molecular studies. LAMP assay proved higher sensitivity than the conventional PCR in detecting parasitemia in early infection with the cystogenic Toxoplasma strain.