Infectious bronchitis (IB) is a highly contagious upper respiratory tract disease of chickens caused by infectious bronchitis virus (IBV), which has various serotypes that do not cross-protect. Vaccine control strategies for this virus are only effective when designed around the currently circulating serotypes. It is essential to not only rapidly detect IBV but also to identify the type of virus causing disease. Six TaqMan™-based quantitative real-time RT-PCR assays (Universal, Ark, Mass, DE/GA98, GA07, GA08) were developed and examined the sensitivity and specificity for each assay. Assays were developed targeting the hypervariable region in the S1 gene subunit. The analytical sensitivity of TaqMan™-based quantitative real-time RT-PCR assays (qRT-PCR) assays was evaluated using synthetic DNA standards that were identical with the target sequence and specificity was further validated using clinical and biological specimens. All developed assays performed equivalently when using synthetic DNA templates as standard material, as it achieved linearity over a 5 log10 dynamic range with a reproducible limit of detection of ≤10 target copies per reaction, with high calculated amplification efficiencies ranging between 90%-115%. Further validation of specificity using clinical and biological specimens was also successful.

中文翻译：

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使用合成DNA标准品和临床标本验证传染性支气管炎的特异性定量实时RT-PCR分析小组。

传染性支气管炎（IB）是由传染性支气管炎病毒（IBV）引起的高度传染性的鸡上呼吸道疾病，它具有多种不能交叉保护的血清型。仅当围绕当前正在传播的血清型进行设计时，针对该病毒的疫苗控制策略才有效。不仅要快速检测IBV，而且要鉴定引起疾病的病毒类型，这一点至关重要。开发了六种基于TaqMan™的定量实时RT-PCR测定法（Universal，Ark，Mass，DE / GA98，GA07，GA08），并检查了每种测定法的敏感性和特异性。针对S1基因亚基中的高变区开发了检测方法。基于TaqMan™的定量实时RT-PCR分析（qRT-PCR）分析的分析灵敏度使用与靶序列相同的合成DNA标准进行评估，并且使用临床和生物学标本进一步验证了特异性。当使用合成DNA模板作为标准材料时，所有已开发的测定均能等效地进行，因为它在5 log10的动态范围内实现了线性，每个反应的检测≤10个目标拷贝的可重复性极限，计算的高扩增效率在90％-115％之间。使用临床和生物学标本对特异性的进一步验证也是成功的。因为它在5 log10的动态范围内实现了线性，每个反应的检出限为≤10个目标拷贝，并且可计算的高扩增效率在90％-115％之间。使用临床和生物学标本对特异性的进一步验证也是成功的。因为它在5 log10的动态范围内实现了线性，每个反应的检出限为≤10个目标拷贝，并且可计算的高扩增效率在90％-115％之间。使用临床和生物学标本对特异性的进一步验证也是成功的。