Real-time PCR to assess the Leishmania load in Lutzomyia longipalpis sand flies: Screening of target genes and assessment of quantitative methods
Real-time PCR to assess the Leishmania load in Lutzomyia longipalpis sand flies: Screening of target genes and assessment of quantitative methods
复制标题
DOI：
10.1016/j.exppara.2011.08.010
复制
发表时间：
2011-11-01
影响因子：
2.1
通讯作者：
Bevilaqua, Claudia M. L.
中科院分区：
文献类型：
--
作者：
Bezerra-Vasconcelos, Diana R.;Melo, Luciana M.;Bevilaqua, Claudia M. L.
内脏利什曼病是巴西的一种地方性疾病，由查加斯婴儿利什曼原虫引起，其主要媒介物种是沙蝇Lutzomyia longipalpis。流行病学研究使用传统的PCR技术来测量在现场收集的白蛉的感染率。然而，实时PCR可以检测较低的寄生虫负荷，减少假阴性的数量，提高白蛉中利什曼原虫寄生虫的定量。本研究比较了不同拷贝数的基因，以检测和定量L. longipalpis标本进行实时PCR。我们将1，10和30只雄性沙蝇与不同数量的水混合在一起。infantum chagasi，形成具有50、500、5000和50，000个利什曼原虫寄生虫的群体。为扩大L.使用婴儿恰加斯病DNA、靶向kDNA的引物、聚合酶α和185核糖体亚基。通过绝对和相对定量测量寄生虫。使用扩增kDNA的PCR检测在测试的基因中表现出最高的灵敏度，显示出检测0.004寄生虫的DNA当量的能力。此外，使用这些引物的相对定量更准确和精确。一般来说，用于DNA提取的白蛉的数量不影响利什曼原虫的定量。然而，对于低拷贝的目标，如聚合酶α基因，样品中较低的寄生虫数量产生不准确的定量。因此，L.通过靶向高拷贝数基因，提高了长须乳杆菌中婴儿恰加斯的扩增;高拷贝数靶点的扩增提高了基于DNA的寄生虫计数的灵敏度、准确度和精密度。(C)2011 Elsevier Inc. All rights reserved.
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 oft. 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 alpha and the 185 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 alpha 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. (C) 2011 Elsevier Inc. All rights reserved.