發(fā)布時(shí)間：2018-02-15 08:23

  本文關(guān)鍵詞： 細(xì)粒棘球絳蟲(chóng) 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(guó)寄生蟲(chóng)學(xué)與寄生蟲(chóng)病雜志》2017年02期 　論文類(lèi)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(chóng)(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(chóng)線(xiàn)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(cè)方法,采用LAMP法和常規(guī)PCR法檢測(cè)細(xì)粒棘球絳蟲(chóng)、泡狀帶絳蟲(chóng)(Cysticercus tenuicollis)、擴(kuò)展莫尼茨絳蟲(chóng)(Moniezia expansa)、羊曲子宮絳蟲(chóng)(Thysaniezia ovilla)、中點(diǎn)無(wú)卵黃腺絳蟲(chóng)(Avitellina centripunctata)、鞭毛線(xiàn)蟲(chóng)(flagella nematodes)、肝片吸蟲(chóng)(Fasciola hepatica)、多房棘球絳蟲(chóng)(E.multilocularis)和捻轉(zhuǎn)血矛線(xiàn)蟲(chóng)(Haemonchus contortus)等9種寄生蟲(chóng)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(cè)梯度稀釋的含cox2基因片段的細(xì)粒棘球絳蟲(chóng)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(cè)50份犬糞樣,計(jì)算細(xì)粒棘球絳蟲(chóng)cox2基因的陽(yáng)性率,評(píng)價(jià)所建立的LAMP法檢測(cè)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(chóng)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(chóng)標(biāo)準(zhǔn)質(zhì)粒濃度為16.09 ag/μl時(shí)仍可見(jiàn)梯狀條帶,而常規(guī)PCR法僅可檢測(cè)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(duì)50份犬糞中細(xì)粒棘球絳蟲(chóng)DNA的檢測(cè)結(jié)果顯示,PCR、LAMP和夾心ELISA法檢測(cè)結(jié)果相同,陽(yáng)性率為8.0%(4/50)。結(jié)論基于細(xì)粒棘球絳蟲(chóng)線(xiàn)粒體cox2基因的LAMP檢測(cè)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(chóng)感染犬的快速檢測(cè)。
[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(guó)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(guó)際科技合作項(xiàng)目(No.2013BC001)~~
【分類(lèi)號(hào)】：R440;R532.32

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