隐孢子虫三种实时荧光定量PCR检测方法的比较

发布时间：2018-04-01 10:15

本文选题：隐孢子虫　切入点：实时荧光定量PCR　出处：《中国动物传染病学报》2017年06期

【摘要】：为筛选一种检测谱广、敏感性好、特异性高的隐孢子虫实时荧光定量PCR法,对JVAP18S法、Csp18S法和CRU18S法三种隐孢子虫实时荧光定量PCR方法的敏感性、特异性和重复性进行了检测,并与基于18S r DNA序列的套式PCR方法进行了比较。结果显示,以10倍倍比稀释的含有微小隐孢子虫18S r DNA基因片段的重组质粒为模板,成功地建立了三种实时荧光定量PCR方法;敏感性试验显示,JVAP18S法和Csp18S法最低可检测0.1个卵囊,而CRU18S法最低只能检测到1个卵囊,但他们均比套式PCR方法敏感;特异性试验结果显示,三种实时荧光定量PCR法均可检测微小隐孢子虫(Cryptosporidium parvum)、泰泽隐孢子虫(Cryptosporidium tyzzeri)和贝氏隐孢子虫(Cryptosporidium baileyi),而其他属寄生虫和细菌DNA的检测结果均为阴性;重复性试验结果显示,JVAP18S法、Csp18S法和CRU18S法的批内变异系数分别为0.69%~2.68%、0.11%~4.93%、0.04%~2.89%,批间变异系数分别为0.52%~5.75%、2.09%~5.13%、1.15%~3.71%;对50份小鼠田间粪便样品检测结果显示,JVAP18S法检测的阳性率为84.00%,显著高于套式PCR法检测的64.00%的阳性率。上述结果表明,三种实时荧光定量PCR法均能有效检测隐孢子虫,比较而言,JVAP18S法敏感性高、特异性强、重复性好,尤其适合于隐孢子虫含量较少的样品检测。
[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.
【作者单位】：吉林农业大学动物科学技术学院;中国农业科学院上海兽医研究所农业部动物产品质量安全生物性危害因子风险评估实验室(上海)农业部动物寄生虫学重点实验室;
【基金】：上海市科技兴农重点攻关项目[沪农科攻字(2015)第1-10号,(2005)第3-4号] 国家农产品质量安全风险评估项目(GJFP201700703) 宁夏回族自治区科技支撑计划[宁农科(2016)1号] 中央级公益性科研院所基本科研业务费(2016JB13) 中国农业科学院科技创新工程经费资助
【分类号】：S852.723

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