本文關(guān)鍵詞： 細(xì)粒棘球絳蟲 細(xì)胞色素c氧化酶亞基基因 環(huán)介導(dǎo)等溫?cái)U(kuò)增　出處：《中國寄生蟲學(xué)與寄生蟲病雜志》2017年02期 　論文類型：期刊論文

【摘要】：目的建立一種安全、快速、高效的細(xì)粒棘球絳蟲(Echinococcus granulosus)的診斷方法——環(huán)介導(dǎo)等溫?cái)U(kuò)增方法(loop-mediated isothermal amplification,LAMP)。方法根據(jù)細(xì)粒棘球絳蟲線粒體細(xì)胞色素c氧化酶亞基2(cytochrome c oxidase subunit 2,cox2)基因序列,設(shè)計(jì)4條LAMP引物,建立LAMP檢測方法,采用LAMP法和常規(guī)PCR法檢測細(xì)粒棘球絳蟲、泡狀帶絳蟲(Cysticercus tenuicollis)、擴(kuò)展莫尼茨絳蟲(Moniezia expansa)、羊曲子宮絳蟲(Thysaniezia ovilla)、中點(diǎn)無卵黃腺絳蟲(Avitellina centripunctata)、鞭毛線蟲(flagella nematodes)、肝片吸蟲(Fasciola hepatica)、多房棘球絳蟲(E.multilocularis)和捻轉(zhuǎn)血矛線蟲(Haemonchus contortus)等9種寄生蟲的DNA以驗(yàn)證LAMP法的特異性;分別用LAMP法和常規(guī)PCR法檢測梯度稀釋的含cox2基因片段的細(xì)粒棘球絳蟲標(biāo)準(zhǔn)質(zhì)粒后,比較兩者的敏感性。采用LAMP、PCR和夾心ELISA法檢測50份犬糞樣,計(jì)算細(xì)粒棘球絳蟲cox2基因的陽性率,評價(jià)所建立的LAMP法檢測效果。結(jié)果LAMP法的特異性驗(yàn)證結(jié)果表明,僅在以細(xì)粒棘球絳蟲DNA為模板的反應(yīng)體系中出現(xiàn)特異性產(chǎn)物。LAMP法在細(xì)粒棘球絳蟲標(biāo)準(zhǔn)質(zhì)粒濃度為16.09 ag/μl時(shí)仍可見梯狀條帶,而常規(guī)PCR法僅可檢測到濃度為16.9 fg/μl以上的質(zhì)粒,LAMP法靈敏性是常規(guī)PCR法的1 000倍。對50份犬糞中細(xì)粒棘球絳蟲DNA的檢測結(jié)果顯示,PCR、LAMP和夾心ELISA法檢測結(jié)果相同,陽性率為8.0%(4/50)。結(jié)論基于細(xì)粒棘球絳蟲線粒體cox2基因的LAMP檢測方法操作方便、特異性較強(qiáng)、敏感性較高,適于細(xì)粒棘球絳蟲感染犬的快速檢測。
[Abstract]:Objective to establish a safe, rapid and efficient method for the diagnosis of Echinococcus granulosus (Echinococcus granulosus) by loop-mediated isothermal amplification. Methods according to the sequence of cytochrome c oxidase subunit 2 cytochrome oxidase subunit 2cox2 of Echinococcus granulosus, Four LAMP primers were designed and LAMP detection method was established. Echinococcus granulosus was detected by LAMP method and routine PCR method. Nine species of DNA parasites, such as Cysticercus tenuicollisa, Moniezia expansa, Thysaniezia ovillailla, Avitellina centripunctata, flagella nematodesa, Fasciola sinensis, E. multilocularisia and Haemonchus contortus, et al. To verify the specificity of LAMP method; The sensitivity of standard plasmid of Echinococcus granulosus containing cox2 gene fragment in gradient dilution was detected by LAMP assay and conventional PCR method respectively. The positive rate of cox2 gene in Echinococcus granulosus was calculated by Lamp PCR and sandwich ELISA method. Results the specificity of LAMP method showed that, Only in the reaction system using Echinococcus granulosus DNA as template was the specific product. Lamp method could still show ladder bands when the standard plasmid concentration of Echinococcus granulosus was 16.09 g / 渭 l. The sensitivity of plasmid lamp assay with concentration above 16.9 fg / 渭 l was 1 000 times higher than that of conventional PCR method. The results of DNA detection for Echinococcus granulosus in 50 dog feces were the same as those detected by sandwich ELISA method. Conclusion the LAMP detection method based on the mitochondrial cox2 gene of Echinococcus granulosus is convenient, specific and sensitive. It is suitable for rapid detection of Echinococcus granulosus infected dogs.
【作者單位】： 新疆農(nóng)墾科學(xué)院 省部共建綿羊遺傳改良與健康養(yǎng)殖國家重點(diǎn)實(shí)驗(yàn)室;石河子大學(xué);
【基金】：新疆生產(chǎn)建設(shè)兵團(tuán)科技創(chuàng)新團(tuán)隊(duì)(No.2014CC004);新疆生產(chǎn)建設(shè)兵團(tuán)科技攻關(guān)項(xiàng)目(No.2014BA002);新疆生產(chǎn)建設(shè)兵團(tuán)國際科技合作項(xiàng)目(No.2013BC001)~~
【分類號(hào)】：R440;R532.32

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