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Assessment of self‐produced PCR methods for the detection of Toxoplasma gondii DNA in meat

Jiang, Shoufu, He, Yanyan, Zhang, Yaoguang, Zhang, Xiaoping, Jiang, Li, Wang, Zhenyu, Zhu, Qian, Ma, Xiaojiang
Journal of food safety 2017 v.37 no.3
DNA, Toxoplasma gondii, bioassays, bradyzoites, fluorescence, fluorescent dyes, genes, meat, monitoring, nucleotide sequences, parasites, pathogens, quantitative polymerase chain reaction, toxoplasmosis
The objective was to develop a highly sensitive and specific polymerase chain reaction (PCR) method for the detection of Toxoplasma gondii DNA in meat. To increase the capacity for the processing of meat samples by improving DNA extraction, four PCR assays were developed and compared using the B1 gene as the target gene for the detection of T. gondii DNA in meat. The results show high specificity and reproducibility for all four PCR assays in clearly detecting DNA samples from four strains of T. gondii while producing no amplification signal for ten other parasites or in healthy meat. Three real‐time quantitative PCR assays were 10‐fold more sensitive than the conventional PCR assay, with a detection limit of 4.5 × 10¹ copies of T. gondii DNA being equivalent to 1.3 bradyzoites. The most sensitive method was SYBR Green I qPCR because it uses special fluorescent dyes directly binding to the amplified double‐stranded DNA sequences and the fluorescence signal is proportional to the amount of PCR products that observed by nearly 1,000‐fold increase in fluorescence intensity. This assay has another advantages over other PCR assays, including less expense (without probe), ease of operation and quicker operation of the test. PRACTICAL APPLICATIONS: The traditional method for detecting T. gondii in meat samples involves the use of a bioassay that is laborious, time‐consuming and not sensitive. The SYBR Green I qPCR assay developed in this study provides a rapid, sensitive, and specific technique and is applicable to surveillance measures for foodborne toxoplasmosis pathogens in meat or meat products as an alternative detection method.