Ashraf Aqeela, Imran Muhammad, Yaqub Tahir, Tayyab Muhammad, Shehzad Wasim, Mingala Claro N, Chang Yung-Fu
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan.
Folia Microbiol (Praha). 2018 May;63(3):373-380. doi: 10.1007/s12223-017-0576-x. Epub 2017 Dec 14.
Mycoplasma mastitis is often difficult to control due to a lack of rapid and accurate diagnostic tools. The aim of the current study was to develop a loop-mediated isothermal amplification (LAMP) assay for the detection of Mycoplasma bovis (M. bovis) in mastitic milk. The assay was developed using primers designed for three different target genes: uvrC, 16S rRNA, and gyrB, and validated using mastitic milk samples previously found positive for the target pathogen. Specificity of the developed assay was determined by testing cross-reactivity of LAMP primers against closely related bovine mastitis bacterial pathogens. The sensitivity was found to be higher compared to conventional polymerase chain reaction (PCR). The LAMP assay was also capable of detecting M. bovis in PCR-negative milk samples of cows with clinical mastitis. The uvrC primers were found to be more sensitive, while gyrB primers were more specific; however, 16S rRNA primers were less specific and sensitive compared to either uvrC or gyrB primers. Cohen's kappa values for uvrC, gyrB, and 16S rRNA primers used in the LAMP assays were 0.940, 0.970, and 0.807, respectively. There was a high level of agreement between the test results and the true-disease status as indicated by the receiver operating characteristic (ROC) curve. Our findings suggest that the newly developed LAMP assays targeting the uvrC and gyrB genes could be a useful tool for rapid and accurate diagnosis of mastitis caused by M. bovis.
由于缺乏快速准确的诊断工具，支原体乳腺炎往往难以控制。本研究的目的是开发一种环介导等温扩增（LAMP）检测方法，用于检测患乳腺炎奶牛乳汁中的牛支原体（M. bovis）。该检测方法使用针对三个不同靶基因（uvrC、16S rRNA和gyrB）设计的引物进行开发，并使用先前检测出目标病原体呈阳性的患乳腺炎乳汁样本进行验证。通过测试LAMP引物与密切相关的牛乳腺炎细菌病原体的交叉反应性，确定所开发检测方法的特异性。结果发现，与传统聚合酶链反应（PCR）相比，该方法的灵敏度更高。LAMP检测方法还能够检测临床型乳腺炎奶牛PCR阴性乳汁样本中的牛支原体。发现uvrC引物更灵敏，而gyrB引物更具特异性；然而，与uvrC或gyrB引物相比，16S rRNA引物的特异性和灵敏度较低。LAMP检测中使用的uvrC、gyrB和16S rRNA引物的Cohen's kappa值分别为0.940、0.970和0.807。如受试者工作特征（ROC）曲线所示，检测结果与真实疾病状态之间具有高度一致性。我们的研究结果表明，新开发的针对uvrC和gyrB基因的LAMP检测方法可能是快速准确诊断牛支原体引起的乳腺炎的有用工具。
