title: Evaluation of a real-time PCR assay based on the single-copy SAG1 gene for the detection of Toxoplasma gondii.
abstract: Real-time PCR-based detection of Toxoplasma gondii is very sensitive and convenient for diagnosing toxoplasmosis. However, the performance of the PCR assays could be influenced by the target gene chosen. Here we evaluate a real-time PCR assay using double-stranded DNA dyes (SYBR(®) Green I assay) with a new set of primers targeting the SAG1 gene for the fast and specific detection of T. gondii. The assay showed higher sensitivity than conventional PCR protocols using T. gondii DNA as template. The detection limit of the developed real-time PCR assay was in the order of 1 tachyzoite. The assay was also assessed by experimentally infected mice and showed positive results for blood (25%), spleen (50%) and lung (50%) as early as 1 dpi. The specificity of the assay was confirmed by using DNA from Neospora caninum, Escherichia coli, Babesia bovis, Trypanosoma brucei, Cryptosporidium parvum, and Toxocara canis. Assay applicability was successfully tested in blood samples collected from slaughtered pigs. These results indicate that, based on SYBR(®) green I, the quantitative SAG1 assay may also be useful in the study of the pathogenicity, immunoprophylaxis, and treatment of T. gondii.
authors.affiliation: Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Haijie
authors.initials: H
authors.lastname: Yu
authors.forename: Bin
authors.initials: B
authors.lastname: Huang
authors.forename: Xunhui
authors.initials: X
authors.lastname: Zhuo
authors.forename: Xueqiu
authors.initials: X
authors.lastname: Chen
authors.forename: Aifang
authors.initials: A
authors.lastname: Du
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016054:DNA, Protozoan / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation; D051379:Mice; D008813:Mice, Inbred ICR; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000953:Antigens, Protozoan; D016054:DNA, Protozoan; D015800:Protozoan Proteins; C047168:SAG1 antigen, Toxoplasma
keywords: Real-time PCR; SAG1; SYBR(®) Green I; Toxoplasma gondii