实时 PCR 和巢式 PCR 在弓形虫视网膜炎患者中的诊断比较。
Comparison of real-time PCR and nested PCR for toxoplasmosis diagnosis in toxoplasmic retinochoroiditis patients.
机构信息
Research Center of Pediatric Infectious Diseases, Institute of Immunology and Infectious Diseases, Iran University of Medical Sciences, Tehran, Iran.
Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
出版信息
BMC Infect Dis. 2021 Nov 23;21(1):1180. doi: 10.1186/s12879-021-06873-3.
BACKGROUNDS
PCR is a proper technique that significantly improves toxoplasmosis diagnosis. However, a more sensitive technique is required. This study compared real-time PCR with nested PCR using B1, SAG-4, and MAG-1 bradyzoite genes to diagnose toxoplasmosis in toxoplasmic retinochoroiditis patients.
METHODS
Blood samples were collected from 10 patients with active toxoplasmic chorioretinal lesions and 10 healthy individuals. Blood samples including peripheral blood mononuclear cells (PBMCs), serum and whole blood samples were used for DNA extraction. Serum was also used to detect anti-toxoplasma IgG and IgM antibodies. Nested PCR and real-time PCR were performed using B1, SAG-4, and MAG-1 target genes.
RESULTS
Five (50%) out of the 10 patients were tested positive for toxoplasmosis with nested PCR using the PBMC samples. All the five patients tested positive with nested PCR were also tested positive for toxoplasmosis with real-time PCR using the PBMC samples. The real-time PCR results demonstrated that 9(90%) out of the 10 patients were positive based on B1 and the remaining one (10%) was positive only based on MAG-1. In general, of the patients, five (50%) were positive using SAG-4 and three (30%) were positive in term of MAG-1 using PBMCs with real-time PCR.
CONCLUSION
It appears that PBMC samples have the best performance as the PCR extraction method and are a good source for toxoplasmosis diagnosis. The use of B22 and B23 target genes due to their high sensitivity and specificity along with bradyzoite genes are recommended for toxoplasmosis diagnosis using PBMC samples with real-time PCR.
背景
PCR 是一种提高弓形虫病诊断的合适技术。然而，需要一种更敏感的技术。本研究比较了使用 B1、SAG-4 和 MAG-1 缓殖子基因的实时 PCR 和巢式 PCR ，以诊断弓形虫性脉络膜视网膜炎患者的弓形虫病。
方法
采集 10 例活动性弓形虫性脉络膜视网膜病变患者和 10 例健康个体的血样。采集外周血单核细胞（PBMC）、血清和全血样本进行 DNA 提取。血清也用于检测抗弓形虫 IgG 和 IgM 抗体。使用 B1、SAG-4 和 MAG-1 靶基因进行巢式 PCR 和实时 PCR。
结果
使用 PBMC 样本进行巢式 PCR 检测，10 例患者中有 5 例（50%）为弓形虫病阳性。所有用 PBMC 进行巢式 PCR 检测阳性的 5 例患者也用 PBMC 进行实时 PCR 检测阳性。实时 PCR 结果显示，10 例患者中有 9 例（90%）基于 B1 为阳性，而其余 1 例（10%）仅基于 MAG-1 为阳性。总体而言，用 PBMC 进行实时 PCR 检测，SAG-4 阳性的患者有 5 例（50%），MAG-1 阳性的患者有 3 例（30%）。
结论
PBMC 样本作为 PCR 提取方法具有最佳性能，是弓形虫病诊断的良好来源。建议使用 B22 和 B23 靶基因，以及缓殖子基因，用于 PBMC 样本的实时 PCR 诊断。
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