Establishment and Preliminary Application of Multiplex PCR for Mycoplasma bovis, Klebsiella pneumoniae and Bovine infectious bovine rhinotracheitis virus
XING Xiao-Yong1, CHENG Jia-Hai1, BAO Shi-Jun1, HAO Bao-Cheng2, WEN Feng-Qin1, WU Xiao-Chun1, HU Yong-Hao1,*
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 2 Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou 730050, China
Abstract Bovine respiratory disease syndrome (BRDC) is a serious disease of cattle caused by a mixture or secondary infection of bacteria, viruses and environmental factors, which seriously endangers the healthy development of cattle industry. Based on the glycoprotein B (gB) gene of Infectious bovine rhinotracheitis virus (IBRV) in GenBank (GenBank No. NC0847.1), lipoprotein 48 (p48) gene of Mycoplasma bovis in GenBank (GenBank No. DQ020482.1) and Khe gene of Klebsiella pneumoniae in GenBank (GenBank No. Kx842080.1), three pairs of primers were designed to amplify the expected fragments of 727, 421 and 192 bp for Infectious bovine rhinotracheitis virus, Mycoplasma bovis and Klebsiella pneumoniae, respectively. The optimization of annealing temperature and primer concentration in PCR amplification, the specific test and clinical sample detection were carried out, and the multiplex PCR and sample evaluation were established. The results showed that the optimum annealing temperature was 52.7 ℃ in the range of 48~54 ℃, and the optimum primer concentration of IBRV, M. bovis , K. pneumoniae was 1, 1, and 15 mol/L in the range of 1~15 mol/L, and the lowest detection rate of IBRV was 103 TCID50, the lowest detectable amount of K. pneumoniae was 8.1×101 cfu and the lowest detection level of M. bovis was 6.5×101 cfu, and no cross reaction was found with Pasteurella multocida, Salmonella typhimurium, Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Serratia marcescens, Pseudomonas aeruginosa and Bovine diarrhea virus. The results of clinical samples were consistent with the isolation and identification of pathogens. The establishment of multiplex PCR method provides strong technical support for the diagnosis and control of bovine respiratory disease syndrome caused by these three pathogens.
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