Sánchez-Carvajal José María, Vera-Salmoral Eduardo, Huerta Belén, Galán-Relaño Ángela, Ruedas-Torres Inés, Larenas-Muñoz Fernanda, Luque Inmaculada, Carrasco Librado, Gómez-Laguna Jaime
Department of Anatomy and Comparative Pathology and Toxicology, Pathology and Immunology Group (UCO-PIG), Unidad de Investigación Competitiva (UIC) Zoonosis y Enfermedades Emergentes ENZOEM, University of Córdoba, Córdoba, Spain.
Department of Animal Health, Unidad de Investigación Competitiva (UIC) Zoonosis y Enfermedades Emergentes (ENZOEM), University of Córdoba, University of Córdoba, Córdoba, Spain.
Front Cell Infect Microbiol. 2024 Feb 26;14:1349999. doi: 10.3389/fcimb.2024.1349999. eCollection 2024.
Bovine tuberculosis (bTB) caused by complex (MTC) remains a significant concern for public health. Direct real-time PCR and droplet digital PCR (ddPCR) are proposed as alternative tools to enhance diagnostic precision and efficiency. This study aims to assess the diagnostic performance of a ddPCR assay targeting IS for the detection of MTC DNA in both microbiological culture and fresh lymph node (LN) tissue samples obtained from cattle, in comparison with the established reference standard, the microbiological culture followed by real-time PCR.
The fresh LNs (N=100) were collected each from a different cattle carcass at the slaughterhouse. The limit of detection of ddPCR-IS was set to 101 copies per 20 μl reaction.
DdPCR-IS detected 44 out of 49 reference-standard positive samples and yielded negative results in 47 out of 51 reference-standard negative samples, resulting in adjusted sensitivity (Se) and specificity (Sp) of 90.76% [95% confidence interval (CI): 82.58 - 98.96%)], and 100% (95% CI: 100%) respectively. The estimated adjusted false negative rate (FNR) was 9.23% (95% CI: 1.04 - 17.42%) and the false positive rate (FPR) was 0% (95% CI: 0%). When directly applied from fresh bovine LN tissues, ddPCR-IS6110 identified 47 out of 49 reference-standard positive samples as ddPCR-IS6110-positive and 42 out of 51 reference-standard negative samples as ddPCR-IS-negative, resulting in adjusted Se and Sp values of 94.80% [95% (CI): 88.52 - 100%] and 100% (95% CI: 100%), respectively. The adjusted FNR was 5.20% (95% CI: 0 - 11.50%) and the FPR was 0% (95% CI: 0%). Noteworthy, ddPCR-IS disclosed as positive 9 samples negative to reference-standard.
DdPCR-IS proved to be a rapid, highly sensitive, and specific diagnostic tool as an alternative to reference-standard method.
由结核分枝杆菌复合群（MTC）引起的牛结核病（bTB）仍是公共卫生领域的重大关注点。直接实时聚合酶链反应（PCR）和液滴数字PCR（ddPCR）被提议作为提高诊断准确性和效率的替代工具。本研究旨在评估一种针对插入序列（IS）的ddPCR检测方法在检测从牛身上获取的微生物培养物和新鲜淋巴结（LN）组织样本中MTC DNA的诊断性能，并与既定的参考标准——微生物培养后进行实时PCR相比较。
从屠宰场不同牛的胴体上各采集100个新鲜淋巴结。ddPCR-IS的检测限设定为每20微升反应101个拷贝。
ddPCR-IS在49个参考标准阳性样本中检测出44个，在51个参考标准阴性样本中有47个呈阴性结果，调整后的灵敏度（Se）和特异度（Sp）分别为90.76%[95%置信区间（CI）：82.58 - 98.96%]和100%（95% CI：100%）。估计的调整后假阴性率（FNR）为9.23%（95% CI：1.04 - 17.42%），假阳性率（FPR）为0%（95% CI：0%）。当直接应用于新鲜牛LN组织时，ddPCR-IS6110在49个参考标准阳性样本中鉴定出47个为ddPCR-IS6110阳性，在51个参考标准阴性样本中有42个为ddPCR-IS阴性，调整后的Se和Sp值分别为94.80%[95%（CI）：88.52 - 100%]和100%（95% CI：100%）。调整后的FNR为5.20%（95% CI：0 - 11.50%），FPR为0%（95% CI：0%）。值得注意的是，ddPCR-IS将9个参考标准为阴性的样本判定为阳性。
ddPCR-IS被证明是一种快速、高度灵敏且特异的诊断工具，可作为参考标准方法的替代方法。
