Scientia Agricultura Sinica ›› 2026, Vol. 59 ›› Issue (19): 4417-4432.doi: 10.3864/j.issn.0578-1752.2026.19.018

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Development and Application of a Multiplex qPCR Detection Kit for Pathogens Causing Bovine Respiratory Disease Complex

LIU XinZhuo(), WANG XinYu, ZHANG YueWei, WU Hao, GU Yue, WANG Shuang, WU WenXue()   

  1. College of Veterinary Medicine， China Agricultural University, Beijing 100193
  • Received:2025-12-30 Accepted:2026-05-07 Online:2026-10-01 Published:2026-10-10
  • Contact: WU WenXue

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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

Fig. 1

Construction of the background nucleic acid library"

Fig. 2

Primer evaluation"

Table 1

Primer and probe sequences"

病原Pathogen 名称Name 序列(5'→3') Sequence(5'→3') 产物长度Product length (bp)
牛支原体
Mp.Bovis		 MP-F1 GCGCACATAGCTGGATTA 138
MP-R1 GGCAAGCTCTTCATCATTTG
MP-P FAM-TGTTCACCCTTCACCTGTGCCATA-MGB
牛分枝杆菌
Mb. bovis		 MB-F1 CCGAATCCCTTGTGAAGTAG 123
MB-R1 CACGACTACGGCTTGTATG
MB-P HEX-TAATGTGCGAGCTGAGCGATGTCGC-MGB
多杀性巴氏杆菌
P.multocida		 PM-F1 AGGCACCTATCATCTCTCTATC 131
PM-R1 CTAAGGCACTGCCCATTAAG
PM-P TexasRed-ATATCGTTTGCCACGTGCCTTGCT-MGB
肺炎克雷伯菌
K.pneumoniae		 KB-F1 ATCTCCGGCATTCCATTTC 97
KB-R1 CAGGTGGATCACACGATTAAA
KB-P CY5-ACCGTGAAGGTATGGCAAAGCTGT-MGB

Table 2

Primer and probe specificity evaluation results"

病原
Pathogen		 名称
Name		 非特异性序列匹配数量Non-specific sequence match count 引物探针二聚体Tm值 Primer-probe dimer Tm (℃)
阴性样本测序结果
Sequencing data from negative samples		 其他病原核酸序列
Other pathogen nucleic acid sequences		 牛基因组
Bovine
genome		 自身二聚体
Primer self-dimer		 交叉二聚体
Primer cross-dimer
牛支原体
Mp.bovis		 MP-F1 None None None 0.3 2.4-38.4
MP-R1 6 None 8 36.0 0.4-50.7
MP-P None None 3 11.0 0.1-22.7
牛分枝杆菌
Mb. bovis		 MB-F1 None None 4 45.4 0.4-41.7
MB-R1 None None None 29.7 0.5-36.1
MB-P None None None 25.5 3.0-41.7
多杀性
巴氏杆菌
P.multocida		 PM-F1 None None 2 25.6 0.1-48.0
PM-R1 1 None 7 17.0 0.0-50.7
PM-P None None 1 10.9 3.1-40.5
肺炎克雷伯菌
K.pneumoniae		 KB-F1 5 None 3 10.6 3.0-38.8
KB-R1 None None 1 10.1 0.0-48.0
KB-P 1 黄病毒1 Flavivirus 1 2 22.5 4.3-40.5

Fig. 3

Performance verification of reference material A: Homogeneity verification of the reference material; B: Stability testing of the reference material after reconstitution; C: Repeated freeze-thaw testing of the reference material after reconstitution"

Fig. 4

Annealing temperature screening A: Annealing temperature optimization for Mycoplasma bovis fluorescent quantitative PCR;B: Annealing temperature optimization for Mycobacterium bovis fluorescent quantitative PCR; C: Annealing temperature optimization for Pasteurella multocida fluorescent quantitative PCR; D: Annealing temperature optimization for Klebsiella pneumoniae fluorescent quantitative PCR"

Fig. 5

Primer and probe concentration screening A: Optimization of primer and probe concentrations for Mycoplasma bovis fluorescent quantitative PCR; B: Optimization of primer and probe concentrations for Mycobacterium bovis Fluorescent quantitative PCR; C: Optimization of primer and probe concentrations for Pasteurella multocida fluorescent quantitative PCR; D: Optimization of primer and probe concentrations for Klebsiella pneumoniae fluorescent quantitative PCR"

Fig. 6

Establishment of the standard curve for quadruplex fluorescent quantitative PCR A: Establishment of the standard curve for Mycoplasma bovis fluorescent quantitative PCR;B: Establishment of the standard curve for Mycobacterium bovis fluorescent quantitative PCR;C: Establishment of the standard curve for Pasteurella multocida fluorescent quantitative PCR;D: Establishment of the standard curve for Klebsiella pneumoniae fluorescent quantitative PCR"

Fig. 7

Amplification curve for sensitivity of quadruplex fluorescence quantitative PCR A: Sensitivity test for copy number in Mycoplasma bovis fluorescent quantitative PCR;B: Sensitivity test for copy number in Mycobacterium bovis fluorescent quantitative PCR;C: Sensitivity test for copy number in Pasteurella multocida fluorescent quantitative PCR;D: Sensitivity test for copy number in Klebsiella pneumoniae fluorescent quantitative PCR"

Fig. 8

Amplification curve for specificity verification of quadruplex fluorescent quantitative PCR A: Specificity detection for Mycoplasma bovis fluorescent quantitative PCR; B: Specificity detection for Mycobacterium bovis fluorescent quantitative PCR; C: Specificity detection for Pasteurella multocida fluorescent quantitative PCR; D: Specificity detection for Klebsiella pneumoniae fluorescent quantitative PCR"

Table 3

Reproducibility test results of quadruplex fluorescence quantitative PCR"

重复1
Replicate 1		 重复2
Replicate 2		 重复3
Replicate 3		 批内变异
Intra-assay CV (%)		 批间变异
Inter-assay CV (%)
牛支原体
Mp. bovis		 批1 Batch 1 16.84 16.73 17.07 1.04 2.47
批2 Batch 2 17.19 17.44 17.66 1.35
批3 Batch 3 17.90 16.73 17.54 3.44
牛分枝杆菌
Mb.bovis		 批1 Batch 1 17.84 17.90 18.78 2.89 2.64
批2 Batch 2 18.90 18.72 18.21 1.91
批3 Batch 3 18.88 18.65 17.69 3.41
多杀性巴氏杆菌
P. multocida		 批1 Batch 1 16.45 15.72 16.31 2.41 2.40
批2 Batch 1 16.68 16.42 16.67 0.89
批3 Batch 3 15.67 16.55 16.66 3.32
肺炎克雷伯菌
K. pneumoniae		 批1 Batch 1 18.02 18.13 17.85 0.78 2.20
批2 Batch 2 17.35 17.64 18.16 2.33
批3 Batch 3 18.39 17.24 18.13 3.37

Table 4

Primer and probe sequences used in conventional PCR"

病原Pathogen 名称Name 序列(5'→3') Sequence(5'→3')
牛支原体
Mp.Bovis		 MP-F GTGCGAGCTGAGCGATGT
MP-R GCCGTTGTAGGCCACTC
牛分枝杆菌
Mb. bovis		 MB-F TGTGTCTACTAGTCCTGGTGTT
MB-R GCGCCGTCAAAATACTGAAGC
多杀性巴氏杆菌
P.multocida		 PM-F GCTTTGCTTGCCACATTATGTTT
PM-R CGTAAAGCCCCACCATTGTT
肺炎克雷伯菌
K.pneumoniae		 KB-F TCTGAAGTGTCTCATTTTCGGGA
KB-R CATCGTCGGGCCCTTCCA

Table 5

Clinical sample test results"

病原名称Pathogen Name 四重荧光定量PCR检测Quadruple qPCR detection 普通PCR检测Conventional PCR detection
牛支原体Mp.Bovis 20（17.09%） 19 (16.24%)
牛分枝杆菌Mb. bovis 17（14.53%） 15 (12.82%)
多杀性巴氏杆菌P.multocida 8（6.84%） 8 (6.84%)
肺炎克雷伯菌K.pneumoniae 53（45.30%） 49 (41.88%)
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