Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA
Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA
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DOI：
10.1590/s0074-02762006000900021
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发表时间：
2006-10-01
期刊：
影响因子：
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通讯作者：
Abath, Frederico GC
中科院分区：
文献类型：
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作者：
Gomes, Ana Lisa do Vale;Melo, Fábio L;Abath, Frederico GC
本报告描述了一种基于SYBR绿色I的真实的实时聚合酶链反应（PCR）方案，用于ABI Prism 7000仪器上的检测。设计了靶向编码SSU rRNA基因的引物，以在真实的实时定量PCR系统中高特异性地扩增曼氏血吸虫DNA。该系统的寄生虫DNA检测限为10 fg纯化的基因组DNA，这意味着小于一个寄生虫细胞的当量（基因组类似于580 fg DNA）。效率为0.99，相关系数（R-2）为0.97。当使用不同拷贝数的靶扩增子作为标准品时，该测定可检测至少10个拷贝的特异性靶。设计所用引物以扩增106 bp DNA片段（Tm 83 ℃）。该方法对沙门氏菌具有高度特异性。mansoni，并没有认识到DNA密切相关的非寄生吸虫。真实的时间PCR允许准确定量S. mansoni DNA，并且不需要通过凝胶电泳对扩增产物进行耗时的PCR后检测。该测定法可能能够定量S。许多样本中的曼氏菌DNA（以及间接的寄生虫负荷），例如蜗牛组织、患者的血清和粪便以及丝虫感染的水。因此，这些PCR方案有可能用作监测血吸虫传播和定量诊断的工具。人类感染。
This report describes the development of a SYBR Green I based real time polymerase chain reaction (PCR) protocol for detection on the ABI Prism 7000 instrument. Primers targeting the gene encoding the SSU rRNA were designed to amplify with high specificity DNA from Schistosoma mansoni, in a real time quantitative PCR system. The limit of detection of parasite DNA for the system was 10 fg of purfied genomic DNA, that means less than the equivalent to one parasite cell (genome similar to 580 fg DNA). The efficiency was 0.99 and the correlation coefficient (R-2) was 0.97. When different copy numbers of the target amplicon were used as standards, the assay could detect at least 10 copies of the specific target. The primers used were designed to amplify a 106 bp DNA fragment (Tm 83 degrees C). The assay was highly specific for S. mansoni, and did not recognize DNA from closely related non-schistosome trematodes. The real time PCR allowed for accurate quantification of S. mansoni DNA and no time-consuming post-PCR detection of amplification products by gel electrophoresis was required. The assay is potentially able to quantify S. mansoni DNA (and indirectly parasite burden) in a number of samples, such as snail tissue, serum and feces from patients, and cercaria infested water Thus, these PCR protocols have potential to be used as tools for monitoring of schistosome transmission and quantitative diagnosis of human infection.