Development of a SYBR green real-time polymerase chain reaction assay for quantitative detection of Babesia gibsoni (Asian genotype) DNA
Development of a SYBR green real-time polymerase chain reaction assay for quantitative detection of Babesia gibsoni (Asian genotype) DNA
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DOI：
10.1177/104063870501700608
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发表时间：
2005-11-01
影响因子：
1.5
通讯作者：
Higuchi, S
中科院分区：
文献类型：
--
作者：
Matsuu, A;Ono, S;Higuchi, S
一种基于SYBR绿色的实时荧光聚合酶链反应（PCR）检测方法，可灵敏、重现和准确地定量吉氏巴氏杆菌（亚洲基因型）。从感染的狗的外周血中提取DNA。通过绘制标准模板的输入量（用含有p18基因的182个碱基对（bp）的质粒DNA构建）对阈值循环数的曲线来创建标准曲线。曲线显示出宽的动态范围（1，000，000倍输入）和高的相关值（> 0.99）。标准模板的PCR扩增效率与用B从外周血中获得的完整基因组DNA的PCR扩增效率相似。Gibsoni感染检测限为9只/μ l吉氏B感染血。阈值循环的批内和批间变异系数分别为0.70%~ 1.89%和1.18%~ 1.92%。该检测系统被认为是可重复的和准确的定量寄生虫DNA在实验感染的狗和CD比传统的显微镜检查更敏感。
A real-time fluorogenic polymerase chain reaction (PCR) assay based on SYBR green that allows for sensitive, reproducible, and accurate quantification of Babesia gibsoni (Asian genotype). DNA from peripheral blood of infected dogs was developed. Standard curves were created by plotting the input amount of a standard template, constructed with plasmid DNA containing 182 base pairs (bp) of the p18 gene, against threshold cycle numbers. The curves showed a wide dynamic range (1,000,000-fold input) and high correlation values (> 0.99). The PCR amplification efficacy of the standard template was similar to that of intact genomic DNA obtained from peripheral blood with B. gibsoni infection. The detection limit of the assay was 9 parasites/mu l of blood with B gibsoni infection. The intra-assay and interassay coefficients of variation of the threshold cycles ranged from 0.70% to 1.89% and from 1.18% to 1.92%, respectively. This assay system was found to be reproducible and accurate for the quantification of parasite DNA in experimentally infected dogs and far CD more sensitive than traditional microscopic examination.