Triplex PCR using new primers for the detection of Toxoplasma gondii

Abstract

Molecular methods are used increasingly for the detection of Toxoplasma gondii infection. This study developed a rapid, sensitive, and specific conventional triplex PCR for the detection of the B1 gene and ITS1 region of T. gondii using newly designed primers and an internal control based on the Vibrio cholerae HemM gene. The annealing temperature and concentrations of the primers, MgCl(2), and dNTPs were optimized. Two sets of primers (set 1 and 2) were tested, which contained different segments of the T. gondii B1 gene, 529 repeat region and ITS1 region. A series of sensitivity tests were performed using parasite DNA, whole parasites, and spiked human body fluids. Specificity tests were performed using DNA from common protozoa and bacteria. The newly developed assay based on set 2 primers was found to be specific and sensitive. The test was capable of detecting as little as 10 pg T. gondii DNA, 10(4) tachyzoites in spiked body fluids, and T. gondii DNA in the organ tissues of experimentally infected mice. The assay developed in this study will be useful for the laboratory detection of T. gondii infection.

Memory-phenotype CD4 + T cells spontaneously generated under steady-state conditions exert innate T H 1-like effector function

“…For detection of T. gondii , total DNA was isolated from peritoneal cells using a DNeasy Blood & Tissue Kit (Qiagen) (57). Real-time PCR was performed utilizing TaqMan Gene Expression Assays (ThermoFisher Scientific) or FastStart Universal SYBR Green Master (ROX) (Roche).…”

Development of touch down-multiplex PCR for the diagnosis of toxoplasmosis

“…Rahamatullah et al in their study used a similar approach for diagnosis of toxoplasmosis using Vibrio cholerae HemM gene as internal control, which would require V. cholerae DNA spiking that may lead to cross contamination. [3] However, we used the human beta globin gene, which is already present in the human samples. This reduces the chances of any cross contamination and helps us rule out presence of inhibitors or a failure of DNA extraction procedure.…”

“…Conventional methods for diagnosis such as parasite culture and immunofluorescence are not universally available and not sensitive enough while serological tests may falsely be negative in early part of infection and in immunosuppressed individuals. [3] Several molecular methods like PCR, LAMP have also been used for the diagnosis of toxoplasmosis. LAMP suffers from issues of false positivity and an inability to target more than one gene sequences i.e.…”

Recent trends in the diagnosis of toxoplasmosis

“…B1 gene has also been used to detect toxoplasmosis in wild birds (Darwich et al, 2012). Of late, triplex PCR using B1 primers was developed for the detection of toxoplasmosis and it provided some very good results (Rahumatullah et al, 2012).…”