Faculty of Medicine, Department of Medical Microbiology, Ankara Yıldırım Beyazıt University, Ankara, Turkey.
Folia Microbiol (Praha). 2020 Apr;65(2):353-361. doi: 10.1007/s12223-019-00731-1. Epub 2019 Jul 31.
Due to limitations in commercial diagnostic methods, this study aimed to develop a reliable real-time polymerase chain reaction (Rt-PCR) assay for early diagnosis of brucellosis. Optimization of the Rt-PCR method was performed on serum samples spiked by Brucella melitensis with different densities ranging from 10 to 10 colony-forming units (cfu)/mL; each density was prepared in ten samples. The limit of detection was investigated by using Thermo DNA extraction kit with Maxima SYBR Green Rt-PCR and two TaqMan probe-based Rt-PCR protocols performed by QuantiTect and TEMPase multiplex PCR master mixes in two thermal cyclers, which were Rotor-Gene and Bio-Rad. The validation of the optimized protocol was carried on 20 brucellosis-negative samples and 20 samples spiked with B. melitensis by using a combination of Thermo DNA extraction kit with TEMPase PCR master mix. SYBR Green Rt-PCR yielded positive results on all samples having ≥ 10 cfu/mL of B. melitensis in both thermal cyclers. Its limit of detection was 112 DNA copies per reaction. The positivity of both probe-based Rt-PCR protocols was 100% and 80% on the samples having 10 cfu/mL and 10 cfu/mL of B. melitensis, respectively. The limit of detection of probe-based protocols was defined as 4 DNA copies per reaction. The optimized Rt-PCR protocol showed high-level accuracy, precision, specificity, and sensitivity, each having a rate of 100%. The current study indicated that the TaqMan probe-based Rt-PCR protocol optimized and validated with serum samples can be reliably used for early diagnosis of brucellosis.
由于商业诊断方法的局限性，本研究旨在开发一种可靠的实时聚合酶链反应（Rt-PCR）检测方法，用于布鲁氏菌病的早期诊断。通过在不同密度（从 10 到 10 个菌落形成单位（cfu）/mL）的布鲁氏菌 melitensis 血清样本中添加 Brucella melitensis 血清样本，对 Rt-PCR 方法进行了优化；每个密度均由十个样本组成。使用 Thermo DNA 提取试剂盒和两种 TaqMan 探针的 Rt-PCR 协议（由 QuantiTect 和 TEMPase 多重 PCR 主混合物在两个热循环仪（Rotor-Gene 和 Bio-Rad）中进行）来研究检测极限。使用 Thermo DNA 提取试剂盒和 TEMPase PCR 主混合物对 20 份布鲁氏菌病阴性样本和 20 份布鲁氏菌 melitensis 加标样本进行了优化方案的验证。SYBR Green Rt-PCR 在两个热循环仪中，所有具有≥10 cfu/mL 的布鲁氏菌 melitensis 的样本均产生阳性结果。其检测极限为每个反应 112 个 DNA 拷贝。两种探针 Rt-PCR 协议的阳性率分别为 100%和 80%，在具有 10 cfu/mL 和 10 cfu/mL 的布鲁氏菌 melitensis 的样本中。探针 Rt-PCR 协议的检测极限定义为每个反应 4 个 DNA 拷贝。优化的 Rt-PCR 方案显示出高水平的准确性、精密度、特异性和敏感性，每个指标的比率均为 100%。本研究表明，优化和验证血清样本的 TaqMan 探针 Rt-PCR 协议可可靠地用于布鲁氏菌病的早期诊断。
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