嵌合DNA-RNA引物在定量PCR检测犬埃立克体和犬巴贝斯虫中的应用。
Use of chimeric DNA-RNA primers in quantitative PCR for detection of Ehrlichia canis and Babesia canis.
作者信息
Peleg Ofer, Baneth Gad, Eyal Osnat, Inbar Jacob, Harrus Shimon
机构信息
Genaphora Ltd, Tel Aviv 69710, Israel.
出版信息
Appl Environ Microbiol. 2009 Oct;75(19):6393-8. doi: 10.1128/AEM.00720-09. Epub 2009 Jul 24.
Abstract
To overcome the problem of nonspecific by-products in quantitative PCR (qPCR) assays, we constructed DNA-RNA chimeric primers and evaluated their use in the detection and quantification of the Ehrlichia canis 16S rRNA, Babesia canis Hsp70, and canine beta-actin genes. Several RNA bases were incorporated into specific positions in the DNA primers, while no RNA stretches were allowed. qPCR reactions were carried out without preamplification steps. This resulted in decreased formation of undesirable by-products and a 10-fold increase in assay sensitivity.
摘要
为克服定量聚合酶链反应（qPCR）检测中非特异性副产物的问题，我们构建了DNA-RNA嵌合引物，并评估了它们在检测和定量犬埃立克体16S rRNA、犬巴贝斯虫热休克蛋白70（Hsp70）以及犬β-肌动蛋白基因中的应用。将几个RNA碱基掺入DNA引物的特定位置，同时不允许存在RNA片段。qPCR反应无需预扩增步骤即可进行。这减少了不良副产物的形成，并使检测灵敏度提高了10倍。
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