Laboratório de Doenças Parasitárias, Programa de Pós-graduação em Ciências Veterinárias, Universidade Estadual do Ceará, Brazil.
Exp Parasitol. 2011 Nov;129(3):234-9. doi: 10.1016/j.exppara.2011.08.010. Epub 2011 Aug 16.
Visceral Leishmaniasis is an endemic disease in Brazil caused by Leishmania infantum chagasi and its main vector species is the sand fly Lutzomyia longipalpis. Epidemiological studies have used conventional PCR techniques to measure the rate of infection of sand flies collected in the field. However, real-time PCR can detect lower parasite burdens, reducing the number of false negatives and improving the quantification of Leishmania parasites in the sand fly. This study compared genes with various copy numbers to detect and quantify L. infantum chagasi in L. longipalpis specimens by real-time PCR. We mixed pools of 1, 10 and 30 male sand flies with various amounts of L. infantum chagasi, forming groups with 50, 500, 5000 and 50,000 Leishmania parasites. For the amplification of L. infantum chagasi DNA, primers targeting kDNA, polymerase α and the 18S ribosome subunit were employed. Parasites were measured by absolute and relative quantification. PCR detection using the amplification of kDNA exhibited the greatest sensitivity among the genes tested, showing the capacity to detect the DNA equivalent of 0.004 parasites. Additionally, the relative quantification using these primers was more accurate and precise. In general, the number of sand flies used for DNA extraction did not influence Leishmania quantification. However, for low-copy targets, such as the polymerase α gene, lower parasite numbers in the sample produced inaccurate quantifications. Thus, qPCR measurement of L. infantum chagasi in L. longipalpis was improved by targeting high copy-number genes; amplification of high copy-number targets increased the sensitivity, accuracy and precision of DNA-based parasite enumeration.
内脏利什曼病是巴西的一种地方性疾病，由婴儿利什曼原虫引起，其主要传播媒介是沙蝇长刺舌蝇。流行病学研究使用传统的 PCR 技术来测量野外采集的沙蝇的感染率。然而，实时 PCR 可以检测到更低的寄生虫负荷，减少假阴性的数量，并提高沙蝇中利什曼原虫寄生虫的定量。本研究比较了具有不同拷贝数的基因，以通过实时 PCR 检测和定量长刺舌蝇中的婴儿利什曼原虫。我们将 1、10 和 30 只雄性沙蝇混合成不同数量的婴儿利什曼原虫，形成每组含有 50、500、5000 和 50000 只利什曼寄生虫的混合群。为了扩增婴儿利什曼原虫 DNA，使用针对 kDNA、聚合酶α和 18S 核糖体亚基的引物。通过绝对和相对定量来测量寄生虫。在测试的基因中，使用 kDNA 扩增的 PCR 检测显示出最高的灵敏度，能够检测到相当于 0.004 个寄生虫的 DNA。此外，使用这些引物的相对定量更准确和精确。总的来说，用于 DNA 提取的沙蝇数量不会影响利什曼虫的定量。然而，对于低拷贝数的目标，如聚合酶α基因，样本中较低的寄生虫数量会导致不准确的定量。因此，通过靶向高拷贝数基因，提高了在长刺舌蝇中检测婴儿利什曼原虫的 qPCR 测量；扩增高拷贝数目标提高了基于 DNA 的寄生虫计数的灵敏度、准确性和精密度。
