本文選題：隱孢子蟲　切入點：實時熒光定量PCR　出處：《中國動物傳染病學(xué)報》2017年06期

【摘要】：為篩選一種檢測譜廣、敏感性好、特異性高的隱孢子蟲實時熒光定量PCR法,對JVAP18S法、Csp18S法和CRU18S法三種隱孢子蟲實時熒光定量PCR方法的敏感性、特異性和重復(fù)性進(jìn)行了檢測,并與基于18S r DNA序列的套式PCR方法進(jìn)行了比較。結(jié)果顯示,以10倍倍比稀釋的含有微小隱孢子蟲18S r DNA基因片段的重組質(zhì)粒為模板,成功地建立了三種實時熒光定量PCR方法;敏感性試驗顯示,JVAP18S法和Csp18S法最低可檢測0.1個卵囊,而CRU18S法最低只能檢測到1個卵囊,但他們均比套式PCR方法敏感;特異性試驗結(jié)果顯示,三種實時熒光定量PCR法均可檢測微小隱孢子蟲(Cryptosporidium parvum)、泰澤隱孢子蟲(Cryptosporidium tyzzeri)和貝氏隱孢子蟲(Cryptosporidium baileyi),而其他屬寄生蟲和細(xì)菌DNA的檢測結(jié)果均為陰性;重復(fù)性試驗結(jié)果顯示,JVAP18S法、Csp18S法和CRU18S法的批內(nèi)變異系數(shù)分別為0.69%~2.68%、0.11%~4.93%、0.04%~2.89%,批間變異系數(shù)分別為0.52%~5.75%、2.09%~5.13%、1.15%~3.71%;對50份小鼠田間糞便樣品檢測結(jié)果顯示,JVAP18S法檢測的陽性率為84.00%,顯著高于套式PCR法檢測的64.00%的陽性率。上述結(jié)果表明,三種實時熒光定量PCR法均能有效檢測隱孢子蟲,比較而言,JVAP18S法敏感性高、特異性強、重復(fù)性好,尤其適合于隱孢子蟲含量較少的樣品檢測。
[Abstract]:In order to screen a real-time fluorescent quantitative PCR method for Cryptosporidium with wide spectrum, good sensitivity and high specificity, the sensitivity, specificity and repeatability of three real-time fluorescent quantitative PCR methods, JVAP18S method Csp18S and CRU18S method, were detected. Compared with the nested PCR method based on 18s r DNA sequence, three real-time quantitative PCR methods were successfully established using recombinant plasmid containing 18s r DNA gene fragment of Cryptosporidium microphylla as template. Sensitivity test showed that only 0.1 oocysts could be detected by JVAP18S method and Csp18S method, but only one egg sac was detected by CRU18S method, but they were more sensitive than nested PCR method. Three real-time fluorescent quantitative PCR methods could be used to detect Cryptosporidium parvumum, Cryptosporidium tyzzeri and Cryptosporidium Baileyii, but the results of other parasites and bacteria DNA were negative. The results of repeatability test showed that the intra-assay variation coefficients of JVAP18S method and CRU18S method were 0.69 and 2.68, respectively, and 0.111.933,0.04 and 0.04.89, respectively, and the coefficient of variation between batches was 0.52 and 5.752.095.153.71, respectively, and the positive rate of JVAP18S method was 84.00 for 50 mouse fecal samples, which was significantly higher than that for nested method. The positive rate of PCR was 64.00%. The three real-time fluorescent quantitative PCR methods can be used to detect Cryptosporidium effectively. Compared with JVAP18S method, JVAP18S method is highly sensitive, specific and reproducible, especially suitable for the detection of Cryptosporidium samples with less content of Cryptosporidium.
【作者單位】： 吉林農(nóng)業(yè)大學(xué)動物科學(xué)技術(shù)學(xué)院;中國農(nóng)業(yè)科學(xué)院上海獸醫(yī)研究所農(nóng)業(yè)部動物產(chǎn)品質(zhì)量安全生物性危害因子風(fēng)險評估實驗室(上海)農(nóng)業(yè)部動物寄生蟲學(xué)重點實驗室;
【基金】：上海市科技興農(nóng)重點攻關(guān)項目[滬農(nóng)科攻字(2015)第1-10號,(2005)第3-4號] 國家農(nóng)產(chǎn)品質(zhì)量安全風(fēng)險評估項目(GJFP201700703) 寧夏回族自治區(qū)科技支撐計劃[寧農(nóng)科(2016)1號] 中央級公益性科研院所基本科研業(yè)務(wù)費(2016JB13) 中國農(nóng)業(yè)科學(xué)院科技創(chuàng)新工程經(jīng)費資助
【分類號】：S852.723

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