為建立一種快速、敏感、特異的豬弓形蟲檢測方法，根據(jù)弓形蟲保守基因序列，設(shè)計一套特異性引物和FAM熒光素標(biāo)記的MGB探針；通過對 PCR反應(yīng)體系和反應(yīng)條件進行優(yōu)化篩選，建立了豬弓形蟲實時熒光定量PCR檢測方法，并對此PCR檢測方法進行了特異性、敏感性、重復(fù)性試驗；利用所建立的方法對60份疑似弓形蟲感染的臨床樣品進行了檢測。結(jié)果顯示：建立的豬弓形蟲實時熒光定量PCR檢測方法在101~107拷貝/μL模板范圍內(nèi)有很好的線性關(guān)系；對弓形蟲重組陽性質(zhì)粒出現(xiàn)陽性擴增信號，但對陰性對照的水和其他7種病原對照未擴增出特異性曲線；最低檢測模板濃度為10拷貝/μL；自60份疑似豬弓形蟲感染樣品中檢出32份陽性，并且和克隆測序結(jié)果一致。結(jié)果表明，本研究建立的豬弓形蟲實時熒光定量PCR檢測方法可用于豬弓形蟲的快速檢測，從而為豬弓形蟲病的診斷提供了特異、敏感、高通量的方法。
Development and Application of a Real-time PCR Method for Detection of Toxoplasma gondii
In order to establish a rapid，sensitive and specific method for detection of Toxoplasmas gondii（T. gondi），a pair of specific primers and FAM（fluorescein）-labeled MGB probes were designed based on the conserved gene sequences of T. gondii. Followed by optimizing PCR reaction system and conditions，a real-time fluorescent PCR assay was established，and the specificity，sensitivity and reproducibility test were carried out；then 60 clinical samples suspected of T. gondii infection were tested by the established method. The results showed that the method expressed a good linear relationship when the template was within the range of 101–107 copies/μL. Specificity test showed that positive signal was observed only when amplifying the recombinant plasmids of T. gondii，rather than the water of negative contrast or other 7 pathogens；the minimum concentration of detection template was 10 copies/μL；32 out of 60 suspected samples were detected positive，and the result was consistent with that of cloning and sequencing. As a conclusion，the established method in this study could be used for rapid detection of T. gondii，which provided a specific，sensitive and high-throughput method for the identification of toxoplasmosis.
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