Real-Time PCR Assay for detection and quantification of Leishmania: standardization, positive control, validation, and intra-laboratory assay

Manuel Hospinal-Santiani, Carlos Ricardo Soccol, Susan Grace Karp, Eduardo Scopel Ferreira da Costa, Luiz Alberto Junior Letti, Germana Davila dos Santos, Vanete Thomaz Soccol
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Abstract

His study aimed to develop a method to investigate PCR sensitivity for diagnosis and ensure reproducibility for parasite load quantification in tissues based on qPCR. In the first step, genes were selected to quantify the parasite load; then, a standard was developed to quantify the concentration of different Leishmania species. These tools were evaluated in intra-laboratory assays. The sensitivity was determined as 0.01 parasites/μL, and the method was reproducible with 100% concordance among human participants in the intra-laboratory validation study. Furthermore, the results demonstrated the specificity of the method in detecting the genus Leishmania without showing cross-reaction with Trypanosoma cruzi or human DNA.
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利什曼原虫检测和定量的实时PCR分析:标准化、阳性对照、验证和实验室内分析
他的研究旨在开发一种方法来研究PCR诊断的敏感性,并确保基于qPCR的组织中寄生虫负荷定量的重复性。第一步,选择基因来量化寄生虫负荷;然后,制定了一个标准来量化不同利什曼原虫种类的浓度。这些工具在实验室内进行了评估。灵敏度为0.01寄生虫/μL,该方法重复性好,实验室内验证研究的一致性为100%。结果表明,该方法在检测利什曼原虫属时具有特异性,且与克氏锥虫或人DNA无交叉反应。
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