Pan Yao, Yu Qingting, Wang Qi, Li Qiang, Tian Wei, Xin Lingxiang, Hu Xing, Xiao Haiyue, Liu Yuanjie, Zhu Luo Rong Deng, Lan Lan, Zhu Liangquan, Wu Jianping
Animal Husbandry Science Institute of Ganzi Tibetan Autonomous Prefecture, Kangding, China.
China Institute of Veterinary Drug Control, Beijing, China.
Front Cell Infect Microbiol. 2025 Jun 4;15:1599817. doi: 10.3389/fcimb.2025.1599817. eCollection 2025.
Yaks serve as a vital economic and ecological resource in high-altitude regions, but it faces significant health challenges from various pathogens. Among these, and are critical pathogens that contribute to severe diseases.
A duplex real-time fluorescence quantitative PCR assay was developed to simultaneously detect and . The species-specific genes and were selected as target regions for primer and probe design. Following rigorous optimization, a duplex assay was established. Recombinant plasmids were constructed to serve as standards for generating standard curves. The detection thresholds were determined using SPSS statistical analysis and receiver operating characteristic curve methods. Furthermore, the assay's sensitivity, specificity, stability, and clinical applicability were evaluated.
The established assay demonstrated high sensitivity, with detection limits of 100 and 10 copies for pMD-kmt1 and pMD-invA, respectively. No cross-reactivity was observed with six pathogens, including , infectious bovine rhinotracheitis virus and others. The standard curves showed strong linearity, with coefficients of determination of 0.995 and 0.998, and amplification efficiencies of 103.37% and 103.47% for pMD-kmt1 and pMD-invA, respectively. No interference was observed between high- and low-concentration templates during simultaneous detection. The intra- and inter-assay coefficients of variation ranged from 0.23% to 1.51%. Detection thresholds were determined to be cycle threshold values of 41.5 for and 40.0 for . Clinical evaluation was performed on 226 samples collected from yaks in seven counties of Ganzi Prefecture, Sichuan Province, China. The single infection rates of and were 20.35% (46/226) and 38.50% (87/226), respectively, while the co-infection rate was 6.19% (14/226).
This study successfully established a duplex real-time fluorescence PCR assay that enables the simultaneous detection of and with high sensitivity, specificity, and efficiency. The assay offers a reliable and rapid diagnostic tool that is particularly suited for clinical and epidemiological investigations in yak populations.
牦牛是高海拔地区重要的经济和生态资源，但面临着来自各种病原体的重大健康挑战。其中，[病原体名称1]和[病原体名称2]是导致严重疾病的关键病原体。
开发了一种双重实时荧光定量PCR检测方法，用于同时检测[病原体名称1]和[病原体名称2]。选择物种特异性基因[基因名称1]和[基因名称2]作为引物和探针设计的靶区域。经过严格优化，建立了双重检测方法。构建重组质粒作为生成标准曲线的标准品。使用SPSS统计分析和受试者工作特征曲线方法确定检测阈值。此外，评估了该检测方法的灵敏度、特异性、稳定性和临床适用性。
所建立的检测方法显示出高灵敏度，pMD-kmt1和pMD-invA的检测限分别为100拷贝和10拷贝。未观察到与六种病原体（包括[病原体名称3]、传染性牛鼻气管炎病毒等）的交叉反应。标准曲线显示出很强的线性，pMD-kmt1和pMD-invA的决定系数分别为0.995和0.998，扩增效率分别为103.37%和103.47%。在同时检测过程中，高浓度和低浓度模板之间未观察到干扰。批内和批间变异系数范围为0.23%至1.51%。确定[病原体名称1]的检测阈值为41.5的循环阈值，[病原体名称2]的检测阈值为40.0的循环阈值。对从中国四川省甘孜州七个县的牦牛采集的226份样本进行了临床评估。[病原体名称1]和[病原体名称2]的单一感染率分别为20.35%（46/226）和38.50%（87/226），而共感染率为6.19%（14/226）。
本研究成功建立了一种双重实时荧光PCR检测方法，能够同时高灵敏度、特异性和高效地检测[病原体名称1]和[病原体名称2]。该检测方法提供了一种可靠且快速的诊断工具，特别适用于牦牛群体的临床和流行病学调查。
