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      1. 國家獸藥產(chǎn)業(yè)技術(shù)創(chuàng)新聯(lián)盟
        National veterinary drug industry technology innovation alliance
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        豬弓形蟲實時熒光PCR檢測方法的建立與應(yīng)用

        時間:2020-06-04   訪問量:1043

        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(jìn)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(jìn)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(jìn)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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        國家獸藥產(chǎn)業(yè)技術(shù)創(chuàng)新聯(lián)盟
        National veterinary drug industry technology innovation alliance

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