新疆农业大学学报2025，Vol.48Issue(5)：419-425,7.DOI:10.20088/j.cnki.jxau.2025.05.008
羊呼吸道主要病原的四重荧光定量PCR方法的建立
Establishment of a Quadruple Fluorescent Quanti-tative PCR Method for Major Pathogens of Sheep Respiratory Tract
摘要
Abstract
To establish a quadruple real-time fluorescence quantitative PCR method for simultaneous detection of Pasteurella multocida(Pm),Mannheimia haemolytica(Mh),Mycoplasma ovipneumoniae(Mo),and Strepto-coccus pneumoniae(Sp).Primers and probes were designed for the kmt1 gene of Pm,the lktD gene of Mh,the tuf gene of Mo,and the yfhA gene of Sp.A quadruple real-time fluorescent quantitative PCR for simultaneous detection of the above four pathogens was established,and the reaction system was optimized.The method was compared with the landmark PCR(DB62/T 2686-2023、DB51/T 2301-2016、DB51/T 1302-2011、DB 22/T 2567-2016)to detect 60 clinical samples.The sensitivity,specificity and repeatability of the method were veri-fied.The results showed that the correlation coefficients(R2)of the standard curves of the multiplex qPCR were 0.999,0.997,0.999,and 0.999 respectively,and the linear relationship was good.The minimum detection lim-its of the recombinant plasmids of Pm,Mh,Mo and Sp were all 102 copies/μL.The results showed that the specificity of the quadruple qPCR reaction was good.The assay confirmed excellent performance,with repeatabil-ity(all CVs＜1%)and clinical agreement as key metrics.The positive rates of Pm,Mh,Mo and Sp detected by this method were 50.0%,53.3%,11.7%and 45.0%respectively,and the coincidence rate with landmark PCR detection was 100%.In conclusion,this study successfully established a quadruple real-time fluorescent quantita-tive PCR detection system for bacterial respiratory diseases in sheep.This method is highly specific and sensi-tive,and can be used for overall prevention and control of respiratory bacterial diseases in sheep,reducing the economic losses caused by them.关键词
多杀性巴氏杆菌/溶血性曼氏杆菌/绵羊肺炎支原体/肺炎链球菌/TaqMan荧光定量PCRKey words
pasteurella multocida/mannheimia haemolytica/mycoplasma ovipneumoniae/streptococcus pneumoniae/TaqMan fluorescence quantitative PCR分类
农业科技引用本文复制引用
武羊羊,王天旗,应佳炜,卢静,贾舒安,储岳峰,杨莉..羊呼吸道主要病原的四重荧光定量PCR方法的建立[J].新疆农业大学学报,2025,48(5):419-425,7.基金项目
自治区天山创新团队计划项目(2025D14006) （2025D14006）
自治区天山英才青年拔尖托举人才项目(2023TSYCQNTJ0022) （2023TSYCQNTJ0022）
自治区重大科技专项项目(2023A02007-2) （2023A02007-2）
自治区重点研发项目(2024B02011) （2024B02011）
中央政府引导地方科技发展专项基金项目(ZYYD2024CG01) （ZYYD2024CG01）
自治区乡村振兴产业发展科技行动项目(2025NC011) （2025NC011）
新疆农业大学大学生创新项目(dxscx2025129) （dxscx2025129）
