Instituto de Investigaciones en Ingeniería Genética y Biología Molecular Dr. Héctor N. Torres (INGEBI-CONICET), Buenos Aires, Argentina.
PLoS Negl Trop Dis. 2013;7(1):e2000. doi: 10.1371/journal.pntd.0002000. Epub 2013 Jan 17.
The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy.
METHODS/PRINCIPAL FINDINGS: We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject.
CONCLUSIONS/SIGNIFICANCE: The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.
敏感、准确和标准化的实时 PCR 方法对于克氏锥虫定量的分析验证至关重要，可为近期感染的诊断以及治疗效果的监测提供可靠的实验室工具。
方法/主要发现：我们已经标准化和验证了一种基于 TaqMan 技术的多重实时定量 PCR 检测方法（qPCR），旨在定量检测克氏锥虫卫星 DNA 以及内部扩增对照（IAC）。IAC 扩增可排除由于抑制物质或在样品处理过程中 DNA 丢失而导致的假阴性 PCR 结果。该检测方法的检测限（LOD）为 0.70 个寄生虫当量/mL，定量限（LOQ）为 1.53 个寄生虫当量/mL，起始于非煮沸的胍 EDTA 血液，其中添加了克氏锥虫 CL-Brener 库存。该方法用来自不同临床阶段和流行病学情况的恰加斯病患者的血液样本进行了评估：1. 16 名来自口传播暴发的委内瑞拉患者，2. 63 名患有慢性恰加斯病的玻利维亚患者，3. 34 名患有慢性恰加斯病的阿根廷病例，4. 27 名来自血清阳性母亲的新生儿，5. 一名接受了来自感染供体的尸体肾移植的血清阴性受体，后者在移植后感染。
结论/意义：该检测方法的性能参数鼓励将其应用于血清学方法无信息的情况下对克氏锥虫感染的早期评估，例如通过口污染或先天性传播的近期感染，或在接受来自血清阳性供体的器官移植后，以及在病因治疗下监测恰加斯病患者。
