Gadu Eman, Hashish Amro, Shelkamy Mostafa M S, Chaves Maria, Onyekachukwu Osemeke, Srednik Mariela E, Sato Yuko, El-Gazzar Mohamed
Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, IA 50011.
Department of Avian and Rabbit Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt.
Avian Dis. 2025 Apr;68(S1):499-505. doi: 10.1637/aviandiseases-D-24-00066.
Spotty liver disease (SLD) is a reemerging infection caused by species that results in increased mortality and reduced egg production, with increased incidence in cage-free commercial layer hens. Recently, has been identified as a key pathogen responsible for SLD. The laboratory diagnosis of SLD primarily relies on isolating colonies, a process hindered by the bacterium's fastidious nature and the requirement for time-consuming and specialized culture conditions. Molecular diagnosis using quantitative real-time PCR (qPCR) overcomes these limitations and offers a more sensitive and specific alternative. However, the existing qPCR assay using a DNA-binding dye chemistry suffers from nonspecific binding to any double-stranded DNA in the sample, which could potentially lead to an increased incidence of false-positive cases. In this study, we present the development of a more specific TaqMan probe-based qPCR assay targeting the glycerol kinase gene of . This assay demonstrated excellent analytical specificity and sensitivity, with a detection limit of 5 copies/µl and a PCR efficiency of 95.15%. Additionally, it exhibited 100% diagnostic specificity and sensitivity. Furthermore, probe-based PCRs are the most commonly used type of diagnostic PCR assay and are much better suited for routine diagnostics compared to other types of PCRs. In conclusion, the newly developed assay represents a significant advancement in the accurate and efficient diagnosis of SLD caused by directly from clinical samples.
斑点状肝病（SLD）是一种由[某种细菌]引起的再度出现的感染性疾病，会导致死亡率上升和产蛋量下降，在非笼养商业蛋鸡中发病率增加。最近，[某种细菌]已被确定为导致SLD的关键病原体。SLD的实验室诊断主要依赖于分离[某种细菌]菌落，该过程受到该细菌苛求特性以及耗时且特殊的培养条件要求的阻碍。使用定量实时PCR（qPCR）进行分子诊断克服了这些局限性，并提供了一种更灵敏且特异的替代方法。然而，现有的使用DNA结合染料化学方法的qPCR检测存在与样品中任何双链DNA非特异性结合的问题，这可能会导致假阳性病例的发生率增加。在本研究中，我们展示了一种针对[某种细菌]甘油激酶基因的更特异的基于TaqMan探针的qPCR检测方法的开发。该检测方法表现出出色的分析特异性和灵敏度，检测限为5拷贝/微升，PCR效率为95.15%。此外，它还表现出100%的诊断特异性和灵敏度。此外，基于探针的PCR是诊断性PCR检测中最常用的类型，与其他类型的PCR相比，更适合常规诊断。总之，新开发的检测方法代表了在直接从临床样本中准确、高效诊断由[某种细菌]引起的SLD方面的重大进展。
