title: 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.
authors.affiliation: Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia. anirah82@gmail.com
authors.forename: Anizah
authors.initials: A
authors.lastname: Rahumatullah
authors.forename: Boon Yin
authors.initials: BY
authors.lastname: Khoo
authors.forename: Rahmah
authors.initials: R
authors.lastname: Noordin
mesh_terms: D000653:Amniotic Fluid / Q000469:parasitology; D000818:Animals; D001921:Brain / Q000469:parasitology; D017931:DNA Primers / Q000592:standards; D016054:DNA, Protozoan / Q000097:blood / Q000134:cerebrospinal fluid / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D006321:Heart / Q000469:parasitology; D006801:Humans; D007668:Kidney / Q000469:parasitology; D008099:Liver / Q000469:parasitology; D051379:Mice; D008810:Mice, Inbred C57BL; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D011897:Random Allocation; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification*; D014124:Toxoplasmosis, Animal / Q000175:diagnosis* / Q000469:parasitology
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan