Quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time
Quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time
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DOI：
10.1128/jcm.40.4.1194-1198.2002
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发表时间：
2002-04-01
影响因子：
9.4
通讯作者：
Ortega-Mora, LM
中科院分区：
文献类型：
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作者：
Collantes-Fernández, E;Zaballos, A;Ortega-Mora, LM
我们报告的发展，实时定量PCR检测感染宿主组织中的犬新孢子虫。该测定使用双链DNA结合染料SYBR绿色I连续监测产物形成。设计寡核苷酸引物，扩增出与N.犬的开发了一种类似的方法来定量28 S rRNA宿主基因，以比较不同样品的寄生虫负荷，并校正DNA样品中潜在的PCR抑制化合物的存在。线性定量检测范围为6 log，每次测定的计算检出限为10（-1）速殖子，线性良好（R-2 = 0.998）。通过使用密切相关的寄生虫弓形虫的DNA证实了检测特异性。在多种宿主脑组织中成功测试了该技术的适用性：（i）根据组织病理学研究观察到的相容性病变，将流产的牛胎分为阴性或阳性新孢子虫感染动物;（ii）实验感染的BALB/c小鼠，分为三组，接种有或无相容性病变的动物和阴性对照。所有样品还通过ITS 1新孢子虫巢式PCR进行了测试，并且在两种PCR技术之间显示出高度一致性（kappa = 0.86）。该技术代表了一种有用的定量诊断工具，可用于研究新孢子虫感染的致病性、免疫预防和治疗。
We report the development of a real-time PCR assay for the quantitative detection of Neospora caninum in infected host tissues. The assay uses the double-stranded DNA-binding dye SYBR Green I to continuously monitor product formation. Oligonucleotide primers were designed to amplify a 76-bp DNA fragment corresponding to the Nc5 sequence of N. caninum. A similar method was developed to quantify the 28S rRNA host gene in order to compare the parasite load of different samples and to correct for the presence of potential PCR-inhibiting compounds in the DNA samples. A linear quantitative detection range of 6 logs with a calculated detection limit of 10(-1) tachyzoite per assay was observed with excellent linearity (R-2 = 0.998). Assay specificity was confirmed by using DNA from the closely related parasite Toxoplasma gondii. The applicability of the technique was successfully tested in a variety of host brain tissues: (i) aborted bovine fetuses classified into negative or positive Neospora-infected animals according to the observation of compatible lesions by histopathological study and (ii) experimentally infected BALB/c mice, divided into three groups, inoculated animals with or without compatible lesions and negative controls. All samples were also tested by ITS1 Neospora nested PCR and a high degree of agreement was shown between both PCR techniques (kappa = 0.86). This technique represents a useful quantitative diagnostic tool to be used in the study of the pathogenicity, immunoprophylaxis, and treatment of Neospora infection.