Hashish Amro, Sinha Avanti, Sato Yuko, Macedo Nubia R, El-Gazzar Mohamed
Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA.
Agriculture Research Center, National Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Giza 12618, Egypt.
Microorganisms. 2022 Feb 1;10(2):341. doi: 10.3390/microorganisms10020341.
(ORT) has been associated with poultry respiratory disease worldwide. The organism is fastidious and isolation is challenging. One TaqMan real-time PCR (qPCR) assay has been developed for the detection of ORT. However, during validating the ORT qPCR, the assay performance was suboptimal. During the in silico evaluation, deviations from the basic parameters for primers and probes designs (e.g., presence of stable undesirable primer-dimers) were observed. The suboptimal design led to low efficiency and low sensitivity of the assay. Initially, modification on the probe was carried out to improve the performance of the assay. However, the assay's performance (efficiency and sensitivity) was still suboptimal. In this manuscript, we describe the development of a new qPCR assay and the comparison of its performance with the currently available assay. A highly efficient, sensitive, and specific qPCR assay was developed with approximately 1000-folds reduction in the limit of detection (from 3 × 10 plasmid DNA copies/mL to 1 × 10 plasmid DNA copies/mL). Additionally, the efficiency of the new assay (E = 98.70%) was significantly better than the current assay (E = 73.18%). The newly developed assay is an improved diagnostic tool for the sensitive and efficient diagnosis of ORT from clinical samples.
禽网状内皮组织增殖症（ORT）在全球范围内都与家禽呼吸道疾病有关。该病原体苛求营养，分离培养具有挑战性。已开发出一种TaqMan实时荧光定量PCR（qPCR）检测方法用于检测ORT。然而，在验证ORT qPCR检测方法时，其检测性能并不理想。在计算机模拟评估过程中，观察到引物和探针设计的基本参数存在偏差（例如，存在稳定的不良引物二聚体）。这种不理想的设计导致该检测方法效率低且灵敏度低。最初，对探针进行了修改以提高检测方法的性能。然而，该检测方法的性能（效率和灵敏度）仍然不理想。在本论文中，我们描述了一种新的qPCR检测方法的开发及其与现有检测方法的性能比较。开发出了一种高效、灵敏且特异的qPCR检测方法，检测限降低了约1000倍（从3×10质粒DNA拷贝/毫升降至1×10质粒DNA拷贝/毫升）。此外，新检测方法的效率（E = 98.70%）明显优于现有检测方法（E = 73.18%）。新开发的检测方法是一种改进的诊断工具，可用于从临床样本中灵敏且高效地诊断ORT。
