Huang Li, Xie Zhixun, Xie Liji, Deng Xianwen, Xie Zhiqin, Luo Sisi, Huang Jiaoling, Zeng Tingting, Feng Jiaxun
Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, 530001, PR China.
College of Life Science and Technology, Guangxi University, Nanning, 530004, PR China.
Virol J. 2015 Feb 12;12:22. doi: 10.1186/s12985-015-0255-y.
Infectious arthritis in broilers represents an economic and health problem, resulting in severe losses due to retarded growth and downgrading at the slaughterhouse. The most common agents associated with cases of infectious arthritis in poultry are avian reovirus (ARV) and Mycoplasma synoviae (MS). The accurate differentiation and rapid diagnosis of ARV and MS are essential prerequisites for the effective control and prevention of these avian pathogens in poultry flocks. This study thus aimed to develop and validate a duplex real-time PCR assay for the simultaneous detection and quantification of ARV and MS.
Specific primers and probes for each pathogen were designed to target the special sequence of the ARV σC gene or the MS phase-variable surface lipoprotein hemagglutinin (vlhA) gene. A duplex real-time PCR assay was developed, and the reaction conditions were optimized for the rapid detection and quantification of ARV and MS.
The duplex real-time PCR assay was capable of ARV- and MS-specific detection without cross-reaction with other non-targeted avian pathogens. The sensitivity of this assay was 2 × 10(1) copies for a recombinant plasmid containing ARV σC or MS vlhA gene, and 100 times higher than that of conventional PCR. This newly developed PCR assay was also reproducible and stable. All tested field samples of ARV and/or MS were detectable with this duplex real-time PCR assay compared with pathogen isolation and identification as well as serological tests.
This duplex real-time PCR assay is highly specific, sensitive and reproducible and thus could provide a rapid, specific and sensitive diagnostic tool for the simultaneous detection of ARV and MS in poultry flocks. The assay will be useful not only for clinical diagnostics and disease surveillance but also for the efficient control and prevention of ARV and MS infections.
肉鸡感染性关节炎是一个经济和健康问题，由于生长迟缓以及在屠宰场被降级处理，会导致严重损失。与家禽感染性关节炎病例相关的最常见病原体是禽呼肠孤病毒（ARV）和滑膜支原体（MS）。准确区分和快速诊断ARV和MS是在家禽群体中有效控制和预防这些禽病原体的必要前提。因此，本研究旨在开发并验证一种用于同时检测和定量ARV和MS的双重实时荧光定量PCR检测方法。
针对每种病原体设计特异性引物和探针，靶向ARV σC基因或MS相变表面脂蛋白血凝素（vlhA）基因的特定序列。开发了一种双重实时荧光定量PCR检测方法，并优化反应条件以快速检测和定量ARV和MS。
该双重实时荧光定量PCR检测方法能够特异性地检测ARV和MS，与其他非靶向禽病原体无交叉反应。该检测方法对含有ARV σC或MS vlhA基因的重组质粒的灵敏度为2×10(1)拷贝，比传统PCR高100倍。这种新开发的PCR检测方法也具有可重复性和稳定性。与病原体分离鉴定以及血清学检测相比，该双重实时荧光定量PCR检测方法能够检测所有测试的ARV和/或MS的现场样本。
这种双重实时荧光定量PCR检测方法具有高度特异性、灵敏性和可重复性，因此可为在家禽群体中同时检测ARV和MS提供一种快速、特异和灵敏的诊断工具。该检测方法不仅对临床诊断和疾病监测有用，而且对ARV和MS感染的有效控制和预防也有用。
