[Real-time fluorescence quantitative polymerase chain reaction detection for Toxoplasma gondii B1 gene in mice urine].

ABSTRACT

A pair of specific primers and a TaqMan probe were designed based on the sequence of Toxoplasma gondii B1 gene from GenBank database. Total DNA of T. gondii was extracted from fresh mice urine. DNA fragment of B1 gene was amplified by PCR. The PCR product was cloned into pMD18-T vector. Following identification, the positive recombinant plasmid was used as reference template to generate standard curve and melt curve. Sensitivity, reproducibility, linear range and stability of reference plasmids were determined. The sensitivity of this method was 10(4) copies/ml. The coefficient of variation (cv) of intra-assay and inter-assay were 2.42% and 4.18%, respectively. Linear range was (10(3)-10(7)) copies/ml. The specificity was 100%. The reference materials were stable. Real-time FQ-PCR of T. gondii DNA in mice urine has been constructed, which is a convenient, sensitive and reliable method for quantifying T. gondii DNA in mice urine.