Tupperwar Nitin, Vineeth Varanasi, Rath Satyajit, Vaidya Tushar
Centre for Cellular and Molecular Biology, Hyderabad 500007, India.
Diagn Microbiol Infect Dis. 2008 May;61(1):23-30. doi: 10.1016/j.diagmicrobio.2007.12.013. Epub 2008 Feb 5.
We have developed a highly accurate and sensitive real-time polymerase chain reaction (PCR) assay to detect and quantify Leishmania parasites. The assay targets GP63, a highly conserved gene in Leishmania. We demonstrate that, with a single assay, we are able to detect and quantify several species of Leishmania. Our assay system detects Leishmania donovani and Leishmania major down to 0.1 parasite. The dynamic range of the assay extends over 6 log cycles of target, with an average correlation coefficient >0.988. In addition, we utilize a simple approach to distinguish between Leishmania species causing diverse spectra of disease. We have also used this assay to follow the course of cutaneous disease in CBA/CaJ mice, known to be resistant to L. major. The assay is sensitive enough to quantify parasite load in the absence of overt lesions and reveals a systemic distribution of Leishmania, which has implications for our understanding of the disease.
我们开发了一种高度准确且灵敏的实时聚合酶链反应（PCR）检测方法，用于检测和定量利什曼原虫寄生虫。该检测方法针对GP63，这是利什曼原虫中一个高度保守的基因。我们证明，通过一次检测，我们能够检测和定量几种利什曼原虫。我们的检测系统能够检测到低至0.1个寄生虫的杜氏利什曼原虫和硕大利什曼原虫。该检测方法的动态范围涵盖目标的6个对数周期，平均相关系数>0.988。此外，我们采用一种简单的方法来区分导致不同疾病谱的利什曼原虫种类。我们还使用该检测方法跟踪已知对硕大利什曼原虫有抗性的CBA/CaJ小鼠的皮肤疾病进程。该检测方法足够灵敏，能够在没有明显病变的情况下定量寄生虫载量，并揭示了利什曼原虫的全身分布，这对我们理解该疾病具有重要意义。
