Due to defects and drawbacks of most conventional diagnostic methods including serology for the diagnosis of toxoplasmosis as a dangerous opportunistic infection in immunocompromised individuals, the accurate, rapid, and sensitive detection of infection in such patients is essential. In this study, the TaqMan probe-based real-time PCR and, a relatively new nucleic acid amplification method, the loop-mediated isothermal amplification (LAMP) technique was compared based on the repetitive elements (RE) sequence to detect Toxoplasma gondii ( T. gondii) DNA in blood samples of immunocompromised individuals. During this study, 119 blood samples from immunocompromised cancer patients with renal failure, undergoing dialysis were studied. After DNA extraction from blood samples using the salt extraction method, the molecular techniques of TaqMan probe-based real-time PCR and LAMP were used to investigate the contamination of the samples with T. gondii, based on the 529 bp (RE) sequence of T. gondii . The analytical sensitivity of LAMP and real-time PCR was evaluated by duplicating the five-step serial dilutions of T. gondii tachyzoites from 0.25 to 5×10 5 spiked tachyzoites per milliliter of the Toxoplasma seronegative blood sample. The extracted DNA from other parasites and human chromosomal DNA were used to determine the specificity of the molecular methods. The obtained results were analyzed using Kappa statistical test and SPSS22 software. Out of 119 studied samples, 7 (5.8%) and 5 (4.2%) samples were positive for Toxoplasma by TaqMan probe-based real-time PCR and LAMP, respectively. The limits of detection of TaqMan probe-based real-time PCR and RE-LAMP in negative serum samples were one and five tachyzoites (CT 38), respectively. Both real-time PCR and LAMP methods were 100% specific for Toxoplasma detection. Positive results were obtained only with T. gondii DNA, while other DNA samples were negative. The TaqMan probe-based real-time PCR based on the RE sequence showed higher sensitivity to T. gondii DNA detection in blood samples of cancer patients and serial dilutions of parasitic tachyzoites. The results show that TaqMan probe-based real-time PRC is a sensitive and specific method for the detection of toxoplasmosis in immunocompromised individuals, as well as the LAMP assay, which can be used as a suitable alternative diagnostic method for the detection of toxoplasmosis in such patients, without need the for any expensive equipment.

中文翻译：

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具有挑战性的基于 TaqMan 探针的实时 PCR 和环介导等温扩增 (LAMP)：用于检测弓形虫病的两种敏感分子技术，弓形虫病是免疫功能低下患者的潜在危险机会性感染

由于包括血清学在内的大多数传统诊断方法的缺陷和缺点，用于将弓形虫病诊断为免疫功能低下个体的危险机会性感染，因此准确、快速和灵敏地检测此类患者的感染至关重要。本研究比较了基于 TaqMan 探针的实时 PCR 和相对较新的核酸扩增方法环介导等温扩增 (LAMP) 技术基于重复元件 (RE) 序列检测弓形虫 (T . gondii) 免疫功能低下个体血液样本中的 DNA。在这项研究中，研究了来自接受透析的免疫功能低下的肾功能衰竭癌症患者的 119 份血液样本。使用盐提法从血样中提取DNA后，根据弓形虫的 529 bp (RE) 序列，使用 TaqMan 基于探针的实时 PCR 和 LAMP 的分子技术研究了弓形虫对样品的污染。LAMP 和实时 PCR 的分析灵敏度通过将弓形虫速殖子的五步连续稀释从每毫升弓形虫血清阴性血样的 0.25 到 5×10 5 掺入的速殖子进行复制来评估。从其他寄生虫中提取的 DNA 和人类染色体 DNA 用于确定分子方法的特异性。所得结果采用Kappa统计检验和SPSS22软件进行分析。在 119 个研究样本中，分别有 7 个 (5.8%) 和 5 个 (4.2%) 样本通过基于 TaqMan 探针的实时 PCR 和 LAMP 呈弓形虫阳性。TaqMan 基于探针的实时 PCR 和 RE-LAMP 在阴性血清样品中的检测限分别为 1 个和 5 个速殖子 (CT 38)。实时 PCR 和 LAMP 方法对弓形虫检测具有 100% 的特异性。仅使用弓形虫 DNA 获得阳性结果，而其他 DNA 样品呈阴性。基于 RE 序列的 TaqMan 探针实时 PCR 对癌症患者血液样本中的弓形虫 DNA 检测和寄生速殖子的连续稀释显示出更高的灵敏度。结果表明，基于 TaqMan 探针的实时PRC 是一种检测免疫功能低下个体弓形体病的灵敏而特异的方法，以及 LAMP 测定法，可作为一种合适的替代诊断方法用于检测免疫功能低下者的弓形体病。这样的患者，