Abdel-Hamid Nour H, Beleta Eman I M, Kelany Mohamed A, Ismail Rania I, Shalaby Nadia A, Khafagi Manal H M
Department of Brucellosis Research, Animal Health Research Institute, Agricultural Research Center, Dokki, Giza 12618, Egypt.
Department of Microbiology, The Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Food, Agricultural Research Center, Dokki, Giza, Egypt.
Vet World. 2021 Jan;14(1):144-154. doi: 10.14202/vetworld.2021.144-154. Epub 2021 Jan 19.
Different polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.
One hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.
TaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.
A cattle serum sample is not the metric of choice for targeting genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between -infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.
不同的聚合酶链反应（PCR）技术已经并仍在用于直接检测不同动物物种和人类血清样本中的DNA，但未经验证或未得到充分验证，导致结果存在差异。因此，本研究旨在评估针对bcsp31基因的TaqMan实时荧光定量PCR（RT-PCR）与传统PCR在准确诊断牛血清中布鲁氏菌属水平布鲁氏菌病方面的诊断性能。
从尼罗河三角洲一些处于检疫措施下的受感染私人农场以及无布鲁氏菌病农场中，收集了184份年龄在1至5岁之间的经细菌学检测为阳性和阴性的奶牛血清样本。这些样本在血清学诊断后分为四组，并通过针对IS711基因的TaqMan RT-PCR和传统PCR进行DNA检测研究。以细菌学检测结果作为金标准，评估两种PCR技术的诊断性能特征。
TaqMan RT-PCR显示出优于传统PCR的性能；它能够分别在属于第1组（血清学和细菌学均为阳性）和第2组（血清学阴性但细菌学阳性）的95%（67/70）和89%（25/28）的牛血清样本中检测到DNA。在评估诊断性能时，TaqMan RT-PCR显示出卓越的诊断敏感性（93.9%）、诊断特异性（88.4%）、性能指数（182.3）、几乎完美的kappa一致性（0.825±0.042）、强正相关（r = 0.826）、基于受试者工作特征（ROC）曲线的高精度以及ROC曲线下面积（0.911），p<0.05且置信区间为95%。
传统PCR并非用于靶向检测牛基因组DNA的首选方法。省时且快速的TaqMan RT-PCR方法在检测牛血清中的DNA时显示出更好的诊断性能。TaqMan RT-PCR所提供的这种性能可被视为朝着使用该技术直接区分感染布鲁氏菌的牛和接种了光滑疫苗的牛迈出的一步，可利用针对此类疫苗的引物从牛血清中进行区分。
