鸽源病毒多重TaqMan实时荧光定量PCR检测方法的建立及应用

Establishment and preliminary application of a multiplex TaqMan real-time quantitative PCR assay for detection of PiAdV, PiCV, and PiHV

摘要:

【背景】 鸽腺病毒(pigeon adenovirus, PiAdV)、鸽圆环病毒(pigeon circovirus, PiCV)与鸽疱疹病毒(pigeon herpesvirus, PiHV)是严重危害鸽群健康的主要病原体，常导致混合感染，给临床鉴别诊断带来困难。【目的】 建立一种可用于同步检测PiAdV、PiCV和PiHV的三重TaqMan荧光定量PCR (real-time quantitative PCR, RT-qPCR)方法。【方法】 通过查找GenBank发布的PiAdV、PiCV和PiHV基因组并加以比对获取其基因保守区域，在保守区域设计引物、探针和制备质粒标准品pMD19-T-PiAdV Ⅰ、pMD19-T-PiAdV Ⅱ、pMD19-T-PiCV及pMD19-T-PiHV；经反应体系优化，标准曲线绘制，评价方法的特异性、敏感性和重复性，建立可同步检测PiAdV、PiCV和PiHV的三重TaqMan RT-qPCR方法；使用该方法进行临床样本检测以评估其临床适用性。【结果】 该PiAdV、PiCV和PiHV的三重TaqMan RT-qPCR方法标准曲线为y=-3.396x+43.210 (R²=0.998 3)、y=-3.540x+40.540 (R²=0.999 4)和y=-3.280x+38.470 (R²=0.998 4)，线性关系良好；与鸽痘病毒、鸽轮状病毒等鸽子其他常见病原及禽腺病毒4型无交叉反应，特异性强；在敏感性方面，该方法的最低检出限分别为10 copies/μL、10 copies/μL和1 copy/μL，敏感度是常规PCR方法的100-1 000倍，敏感性高；批内、批间重复性试验的变异系数均小于2.0%，重复性和稳定性良好。使用建立的PiAdV、PiCV和PiHV的三重TaqMan RT-qPCR检测方法对152份鸽子临床样本进行检测，RT-qPCR阳性检出率(PiAdV 55.92%、PiCV 51.32%、PiHV 40.13%)略高于常规PCR方法，具有良好的临床适用性。【结论】 本研究建立的PiAdV、PiCV和PiHV的三重TaqMan RT-qPCR检测方法标准曲线的线性关系良好、特异性强、敏感性高、重复性良好，可快速、高效、特异检测临床样本中的PiAdV、PiCV和PiHV，为PiAdV、PiCV和PiHV早期诊断、协同致病机制研究和流行病学调查提供一种高效的技术手段。

Abstract:

[Background] Pigeon adenovirus (PiAdV), pigeon circovirus (PiCV), and pigeon herpesvirus (PiHV) are major pathogens that seriously jeopardize the health of pigeon flocks and often lead to coinfections, posing challenges for clinical differential diagnosis. [Objective] To establish a triplex TaqMan fluorescent quantitative PCR (real-time quantitative PCR, RT-qPCR) method for the simultaneous detection of PiAdV, PiCV, and PiHV. [Methods] We obtained the conserved regions by searching and comparing the genomes of PiAdV, PiCV, and PiHV published in GenBank. Primers and probes were designed based on the conserved region, and plasmid standards pMD19-T-PiAdV Ⅰ, pMD19-T-PiAdV Ⅱ, pMD19-T-PiCV, and pMD19-T-PiHV were prepared. The reaction systems were optimized and the standard curves were drawn to evaluate the specificity, sensitivity, and repeatability of the established assay. A triplex TaqMan RT-qPCR assay for simultaneous detection of PiAdV, PiCV, and PiHV was thus established. It was then used to test the clinical samples, and its clinical applicability was established. [Results] The standard curves of the triple TaqMan RT-qPCR assay for PiAdV, PiCV, and PiHV were y=–3.396x+43.210 (R²=0.998 3), y=–3.540x+40.540 (R²=0.999 4), and y=–3.280x+38.470 (R²=0.998 4), respectively, showing good linear relationships. The assay showed no cross-reaction with other common pigeon pathogens such as pigeon pox virus and pigeon rotavirus, as well as fowl adenovirus type 4, showcasing strong specificity. For PiAdV, PiCV, and PiHV, the assay showed the minimum detection limits of 10 copies/μL, 10 copies/μL, and 1 copy/μL, respectively, which are 100 to 1 000 times more sensitive than the conventional PCR method, indicating high sensitivity. The coefficient of variation was less than 2.0% in both intra-batch and inter-batch repeatability tests, demonstrating good repeatability and stability. The established triple TaqMan RT-qPCR assay for PiAdV, PiCV, and PiHV was used to detect 152 pigeon clinical samples and showed slightly higher positive rates (PiAdV 55.92%, PiCV 51.32%, and PiHV 40.13%) than the conventional PCR method, demonstrating good clinical applicability. [Conclusion] The established triple TaqMan RT-qPCR assay for the detection of PiAdV, PiCV, and PiHV has good linear relationships and high specificity, sensitivity, and repeatability. It can be used for rapid, efficient, and specific detection of PiAdV, PiCV, and PiHV in clinical samples, serving as an efficient technical method for early diagnosis of the three viruses as well as for the study of synergistic pathogenic mechanism and epidemiological investigation.