Establishment and Application of Fluorescent Quantitative PCR Assay for Laboratory Animal Toxoplasma gondii
Objective To establish and apply TaqMan probe real-time fluorescent quantitative PCR method for laboratory animal Toxoplasma gondii.Method By comparing the sequences of various strains of Toxoplasma gondii published by NCBI,the conservative region of 529 bp repetitive sequence was selected to design primers and probe,and a fluorescence quantitative PCR method was established.Subsequently,the specificity and sensitivity of the method was confirmed.Plasmid standards were obtained from 106 to 102 copies/μL in 10-fold series dilution,and 3 parallels were detected for each concentration and repeated 3 times.The coefficient of variation intra-assay and inter-assay was calculated to evaluate the reproducibility and stability of the method.14 canine tissue samples and 66 porcine whole blood samples were detected by the established quantitative PCR method and the PCR method recommended by national standard to evaluate the capability of this method in practical application.Result The established quantitative PCR method showed a linear relationship between Ct value and plasmid concentration in the range of 108～101 copies/μL,the standard curve Slope was-3.285,R2 value was 1,and the amplification efficiency was 101.663%.The lowest detectable concentration of Toxoplasma gondii was 101 copies/μL.There was no cross reaction with other 14 common swine pathogens.Replicates were detected at 106 to 102 copies/μL plasmid standards,and the coefficient of variation intra-assay and inter-assay was less than 1.21%and 0.62%,respectively.The established quantitative PCR method and the national standard PCR method were used to detect the positive rate of 14 canine tissue samples and 66 porcine whole blood samples.The result showed that the positive rate of the established quantitative PCR method and the national standard PCR method were 10%(8/80)and 7.5%(6/80),respectively,and the positive coincidence rate reached 100%.Conclusion The established Toxoplasma gondii FQ-PCR method can effectively detect Toxoplasma gondii.It provides a good method for daily monitoring of toxoplasma gondii in laboratory animals.
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