Development of absolute quantification method for genotype-specific Babesia microti using real-time PCR and practical experimental tips of real-time PCR.
Development of absolute quantification method for genotype-specific Babesia microti using real-time PCR and practical experimental tips of real-time PCR.
复制标题
使用实时 PCR 和实时 PCR 的实用实验技巧开发基因型特异性田鼠巴贝虫的绝对定量方法。
DOI：
10.1016/j.parint.2016.03.003
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
Saito-Ito A.
中科院分区：
文献类型：
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作者：
Ohmori S;Nagano-Fujii M;Saito-Ito A.
田鼠巴贝斯虫（Babesia microti），啮齿类巴贝斯虫的一种，被认为是人畜共患病、人类巴贝斯虫病的病原体，由多种基因型的小亚基核糖体RNA基因（SSUrDNA）组成，不同基因型对人类具有不同的传染性和致病性。我们使用 SYBR Green I 建立了实时 PCR 测定，可对每种 SSUrDNA-B 型进行特异性检测和绝对定量。即使在混合感染中，日本啮齿动物中也发现了四种 SSUrDNA 类型的缩微。在此测定中，使用了四个针对内部转录间隔区 1 或 2 序列的基因型特异性引物对。引物对具有对同源基因型DNA高度特异性的特点。所有四种基因型的循环阈值 (Ct) 值与 DNA 对数浓度的校准曲线在 107 倍 DNA 浓度范围内呈线性，相关系数为 0.95 至 1，并且扩增效率为 90% 至 110%。即使存在异源 DNA，所有四种基因型的标准曲线也不会改变。在本文中，我们介绍了如何建立和执行基因型特异性实时 PCR 以及我们推荐的实用实验技巧。
Babesia microti, a rodent babesia, is known as a pathogen of zoonosis, human babesiosis, is composed of several genotypes of small subunit ribosomal RNA gene (SSUrDNA) and different genotypes have been suggested to have different infectivity and pathogenicity to humans. We established a real-time PCR assay using SYBR Green I, which allows specific detection and absolute quantification for each SSUrDNA-type-B. microtiof four SSUrDNA-types found in Japanese rodents even in mixed infection. In this assay, four genotype-specific primer pairs targeted on internal transcribed spacer 1 or 2 sequences were used. Primer pairs have the characteristics for a high specificity for homologous genotype DNA. The calibration curves of cycle threshold (Ct) values versus log concentrations of DNA for all four genotypes were linear over 107fold range of DNA concentrations with correlation coefficient from 0.95 to 1 and sufficient amplification efficiency from 90% to 110%. The standard curves for all four genotypes were not changed even in the presence of heterologous DNA.In this paper, we introduce how to establish and perform the genotype-specific real-time PCR and our practical experimental tips to be recommended.