Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA.
J Clin Microbiol. 2010 Jul;48(7):2424-32. doi: 10.1128/JCM.02405-09. Epub 2010 May 12.
Insufficient diagnostic sensitivity and specificity coupled with the potential for cross-reactivity among closely related Anaplasma species has made the accurate determination of infection status problematic. A method for the development of simplex and duplex real-time quantitative reverse transcriptase PCR (qRT-PCR) assays for the detection of A. marginale and A. phagocytophilum 16S rRNA in plasma-free bovine peripheral blood samples is described. The duplex assay was able to detect as few as 100 copies of 16S rRNA of both A. marginale and A. phagocytophilum in the same reaction. The ratio of 16S rRNA to 16S DNA copies for A. marginale was determined to be 117.9:1 (95% confidence interval [95% CI], 100.7:1, 135.2:1). Therefore, the detection limit is the minimum infective unit of one A. marginale bacterium. The duplex assay detected nonequivalent molar ratios as high as 100-fold. Additionally, the duplex assay and a competitive enzyme-linked immunosorbent assay (cELISA) were used to screen 237 samples collected from herds in which anaplasmosis was endemic. When the cELISA was evaluated by the results of the qRT-PCR, its sensitivity and specificity for the detection of A. marginale infection were found to be 65.2% (95% CI, 55.3%, 75.1%) and 97.3% (95% CI, 94.7%, 99.9%), respectively. A. phagocytophilum infection was not detected in the samples analyzed. One- and two-way receiver operator characteristic curves were constructed in order to recommend the optimum negative cutoff value for the cELISA. Percentages of inhibition of 20 and 15.3% were recommended for the one- and two-way curves, respectively. In conclusion, the duplex real-time qRT-PCR assay is a highly sensitive and specific diagnostic tool for the accurate and precise detection of A. marginale and A. phagocytophilum infections in cattle.
由于缺乏诊断的敏感性和特异性，并且密切相关的无形体种属之间存在交叉反应，使得准确确定感染状态成为问题。本研究描述了一种用于开发用于检测无浆体游离牛外周血样本中边缘无形体和嗜吞噬细胞无形体 16S rRNA 的单重和双重实时定量逆转录 PCR（qRT-PCR）检测方法。该双重检测法能够在相同反应中检测到低至 100 拷贝的两种无形体的 16S rRNA。确定边缘无形体 16S rRNA 与 16S DNA 拷贝的比例为 117.9:1（95%置信区间[95%CI]，100.7:1，135.2:1）。因此，检测下限是一个边缘无形体细菌的最小感染单位。该双重检测法检测到高达 100 倍的不等摩尔比值。此外，该双重检测法和竞争酶联免疫吸附测定（cELISA）被用于筛查来自地方性无形体病牛群的 237 个样本。当用 qRT-PCR 的结果评估 cELISA 时，发现其检测边缘无形体感染的敏感性和特异性分别为 65.2%（95%CI，55.3%，75.1%）和 97.3%（95%CI，94.7%，99.9%）。在分析的样本中未检测到嗜吞噬细胞无形体感染。为了推荐 cELISA 的最佳阴性截断值，构建了单重和双重接收者操作特征曲线。建议单重和双重曲线的抑制百分比分别为 20%和 15.3%。总之，该双重实时 qRT-PCR 检测法是一种高度敏感和特异的诊断工具，可用于准确和精确地检测牛的边缘无形体和嗜吞噬细胞无形体感染。
