关键词: 牛呼吸道疾病综合征, 多重荧光定量PCR, 病原菌检测, 检测试剂盒

Abstract:

【Background】In recent years, with the continuous growth of China's demand for beef and milk consumption, coupled with the expansion of breeding scale, the need for cattle health monitoring has become increasingly urgent. Bovine respiratory disease complex (BRDC), as one of the most devastating diseases, has shown a significant upward trend in incidence, causing sustained economic losses to the cattle industry. The etiology of BRDC in calves is complex, with frequent co-infections among pathogens, significantly increasing diagnostic difficulty. Current commercial detection kits suffer from limitations such as limited variety and low throughput, failing to meet the demand for early and accurate diagnosis in large-scale farms.【Objective】This study aims to develop a multiplex quantitative real-time PCR (qPCR) kit with high specificity, sensitivity, and reproducibility for the simultaneous detection of four major pathogenic bacteria associated with BRDC, thereby overcoming the long detection cycles and low sensitivity of traditional methods and providing reliable technical support for rapid diagnosis and scientific prevention and control of BRDC.【Method】Nasal swabs were collected from healthy calves rigorously confirmed negative for the four target pathogens. High-throughput sequencing was employed to construct a nasal cavity microecological background nucleic acid database, and total nucleic acids were extracted to prepare "background nucleic acid samples" for simulating the complex interference matrix of clinical specimens. Specific primers and probes were designed targeting conserved gene regions of Mycoplasma bovis, Mycobacterium bovis, Pasteurella multocida, and Klebsiella pneumoniae. A multi-step bioinformatics screening strategy was employed to rigorously evaluate primer secondary structures and their homology with non-target background nucleic acids, thereby eliminating the risk of cross-reactivity with non-target flora. Positive and negative controls containing authentic sample interference components were prepared by mixing background nucleic acid samples with buffer solution and pathogen nucleic acid samples, respectively. The annealing temperature was optimized using gradient settings, primer and probe concentrations were optimized using a matrix method, and the kit's sensitivity, specificity, and reproducibility were systematically evaluated. Validation was performed using 117 clinical samples.【Result】The detection sensitivity of the developed kit reached 1 copy/μL for Mp. bovis and M. bovis, and 10 copies/μL for P. multocida and K. pneumoniae, demonstrating high amplification efficiency. Specificity validation showed no cross-reactivity with common bovine respiratory commensal bacteria or other pathogens, and no non-specific amplification signals were observed. In reproducibility tests, both intra-batch and inter-batch coefficients of variation were both below 10%, indicating good stability and repeatability. The detection rates in clinical samples were 17.09%, 14.53%, 6.84%, and 45.30%, respectively, and all positive samples were confirmed by sequencing.【Conclusion】This study successfully developed a Multiplex quantitative real-time PCR detection kit capable of simultaneously, rapidly, sensitively, and specifically detecting the four major BRDC pathogens: Mp.bovis, Mb.bovis, P.multocida, and K.pneumoniae. By incorporating background nucleic acid simulation and a multi-step bioinformatics screening strategy, this kit effectively addresses the issue of non-specific interference in complex clinical samples, ensuring the accuracy and reliability of the detection system. With its high sensitivity, strong specificity, and good reproducibility, it shortens the detection cycle from several days with traditional methods to within 2 hours, significantly improving clinical diagnostic efficiency. This kit provides key technical support for early rapid diagnosis, epidemic surveillance and warning, and precision medication-based prevention and control, demonstrating broad prospects for application and popularization.

Key words: bovine respiratory disease complex, multiplex quantitative real-time PCR, pathogen detection, detection kit