da Fonseca Flávia Moreira, de Oliveira Koch Marilia, Sato Ana Paula, Rodriguez Maria Constanza, Locatelli-Dittrich Rosangela
Department of Veterinary Medicine, Federal University of Paraná, Rua Dos Funcionários, 1540, Curitiba, Paraná, 80035-050, Brazil.
Veterinary Laboratory, Vetsuisse Fakultät, University of Zurich, Winterthurerstr.260, CH-8057, Zurich, Switzerland.
Acta Parasitol. 2025 Jan 3;70(1):2. doi: 10.1007/s11686-024-00939-3.
The aim of the present study was to establish a SYBR Green-based real-time PCR assay for detection of the Nc5 segment from the Neospora caninum genome.
The oligonucleotides sequences targeting the Nc5 gene previously reported and designed in-house were validated. Two Primer sets were evaluated and tested in four different combinations. The NP7/NP10 assay was selected and reaction conditions optimized. Efficiency, analytical sensitivity, precision and specificity were assessed. The assay was evaluated in triplicate, in three independent PCR runs performed by two technicians to generate robust results.
The standard curve determined by tenfold serial dilutions (1 to 1 × 10-) established a reaction efficiency (E) of 102.34%, a correlation coefficient (R) of 0.999 and a slope of -3.267. LOD of the real-time PCR assay was 0.456 tachyzoites DNA per reaction, as compared to 45.62 for the conventional method. SYBR green real-time PCR was 100 times more sensitive than the conventional method. Precision analysis showed 100% intra- and inter-assay repeatability at the minimum detection limit. The mean assay coefficient of variation (CV%) was 4.19% and standard deviation (SD) 1.67%. No significant differences between the means of Cq in the replicates and technicians (P > 0.05) was found, indicating that the assay is robust and accurate. The applicability of the assay was tested and N. caninum DNA was detected in milk, blood, amniotic fluid, placenta and different tissue samples.
The protocol had high specificity, confirmed by melting curve analysis and no cross-reactions with other tested microorganisms. The SYBR Green-based PCR protocol standardized in this study is a highly sensitive and specific method, reproducible and applicable for the detection of N. caninum in different biological samples.
本研究旨在建立一种基于SYBR Green的实时荧光定量PCR检测方法，用于检测犬新孢子虫基因组中的Nc5片段。
对先前报道的和自行设计的靶向Nc5基因的寡核苷酸序列进行验证。评估了两组引物，并以四种不同组合进行测试。选择了NP7/NP10检测方法并优化了反应条件。评估了效率、分析灵敏度、精密度和特异性。该检测方法由两名技术人员进行三次独立的PCR运行，每次重复三次，以产生可靠的结果。
通过十倍系列稀释（1至1×10-）确定的标准曲线显示反应效率（E）为102.34%，相关系数（R）为0.999，斜率为-3.267。实时荧光定量PCR检测方法的最低检测限为每个反应0.456个速殖子DNA，而传统方法为45.62。SYBR Green实时荧光定量PCR比传统方法灵敏100倍。精密度分析表明，在最低检测限处，批内和批间重复性均为100%。平均检测变异系数（CV%）为4.19%，标准差（SD）为1.67%。在重复样本和技术人员之间未发现Cq平均值的显著差异（P>0.05），表明该检测方法稳健且准确。测试了该检测方法的适用性，在牛奶、血液、羊水、胎盘和不同组织样本中检测到了犬新孢子虫DNA。
通过熔解曲线分析证实该方法具有高特异性，且与其他测试微生物无交叉反应。本研究中标准化的基于SYBR Green的PCR方法是一种高度灵敏和特异的方法，可重复且适用于检测不同生物样本中的犬新孢子虫。
