Bovine tuberculosis (bTB) is an ongoing issue in several countries within the European Union. Microbiological culture is the official confirmation technique for the presence of Mycobacterium tuberculosis complex (MTBC) members in bovine tissues, but several methodological issues, such as moderate sensitivity and long incubation times, require the development of more sensitive and rapid techniques. This study evaluates the analytical and diagnostic performance, comparative to culture, of a real-time PCR targeting the MTBC-specific IS6110 transposon using a panel of bovine tissue samples sourced from the Spanish bTB eradication campaign. Robustness and repeatability were evaluated in an interlaboratory trial between European Union National Reference Laboratories. The limit of detection with 95% confidence was established at 65 fg/reaction of purified genomic equivalents. Diagnostic sensitivity (Se) and specificity (Sp) were, respectively, 96.45% and 93.66%, and the overall agreement (κ) was 0.88. Cross-reactivity was detected against two mycobacterial isolates identified as Mycobacterium marinum and “Mycobacterium avium subsp. hominissuis,” and whole-genome sequencing (WGS) analysis of the latter isolate revealed an IS6110-like sequence with 83% identity. An identical IS-like element was found in other Mycobacterium avium complex species in the NCBI nucleotide and WGS databases. Despite this finding, this methodology is considered a valuable alternative to culture, and the strategy of use should be defined depending on the control or eradication programs.

中文翻译：

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在组织样品上直接PCR检测结核分枝杆菌复合体：细菌培养的替代方法

牛结核病（bTB）在欧盟内部的多个国家中一直是一个持续存在的问题。微生物培养是牛组织中结核分枝杆菌复合物（MTBC）成员存在的官方确认技术，但是一些方法学问题，例如中等敏感性和长孵育时间，要求开发更灵敏和快速的技术。这项研究评估针对MTBC特异性IS 6110的实时PCR与培养相比的分析和诊断性能使用一组来自西班牙bTB根除运动的牛组织样本进行转座子。在欧盟国家参考实验室之间的实验室间试验中评估了鲁棒性和可重复性。纯化的基因组当量的反应浓度为65 fg /反应时，检测限为95％置信度。诊断敏感性（Se）和特异性（Sp）分别为96.45％和93.66％，总体一致性（κ）为0.88。检测到与两种分枝杆菌分离物的交叉反应性，这两种分离物分别为海洋分枝杆菌和鸟分枝杆菌亚种。hominissuis，后者分离的”和全基因组测序（WGS）分析揭示的IS 6110类序列，具有83％的同一性。在NCBI核苷酸和WGS数据库的其他鸟分枝杆菌复杂物种中发现了相同的IS样元素。尽管有这一发现，但该方法仍被认为是替代文化的宝贵方法，应根据控制或根除计划确定使用策略。