副猪嗜血杆菌及巴氏杆菌双重荧光定量PCR检测方法的建立
|
摘要
为建立副猪嗜血杆菌(Hps)、多杀性巴氏杆菌(Pm)的双重荧光定量PCR检测方法,本研究基于Hps的Omp P2基因,Pm的PlpE基因设计两对特异性引物及探针,通过对反应条件优化,建立了一种同时检测Hps及Pm的双重荧光定量PCR方法。该方法能够特异性地检测Hps和Pm,其对重组质粒标准品的最低检测浓度分别为5.60×10~2拷贝/μL、7.58×10~2拷贝/μL。双重与单一荧光定量PCR最低检测限相同,且均是常规PCR的100倍。重复性试验结果显示,该方法的组内和组间变异系数均小于2.5%。临床应用结果显示:该方法对阳性样品的检出率为53.57%,明显优于常规PCR和细菌分离鉴定。该方法能够用于两种疾病的同时检测和快速排查疾病。为两种疾病的防治提供有效检测工具。
In order to establish a duplex fluorescent quantitative PCR assay for Haemophilus parasuis(Hps) and Pasteurella(Pm), two pairs of primers and Taq Man probes were designed according to the conserved sequence of the OmpP2 gene and Plp E gene in this study, through optimizing all components concentration and annealing temperature in the reaction system, an optimal duplex qPCR method was established to simultaneously detect Hps and Pm. This method can specifically amplify OmpP2 gene and PlpE gene, and the limited detection content of plasmid standard could reach about 5.60 ×10~2 copies/μL, 7.58 ×10~2 copies/μL respectively, which were approximately a 10~2-fold greater sensitivity than conventional PCR. The variation coefficients of intra-assay and inter-assay were both less than 2.5%. The results of clinical application showed that the positive rate was 53.57%,which was obiously better than conventional PCR test, bacterial isolation and identification. This method could be used to detect two diseases simultaneously, and it laid the foundation for subsequent treatment.
In order to establish a duplex fluorescent quantitative PCR assay for Haemophilus parasuis(Hps) and Pasteurella(Pm), two pairs of primers and Taq Man probes were designed according to the conserved sequence of the OmpP2 gene and Plp E gene in this study, through optimizing all components concentration and annealing temperature in the reaction system, an optimal duplex qPCR method was established to simultaneously detect Hps and Pm. This method can specifically amplify OmpP2 gene and PlpE gene, and the limited detection content of plasmid standard could reach about 5.60 ×10~2 copies/μL, 7.58 ×10~2 copies/μL respectively, which were approximately a 10~2-fold greater sensitivity than conventional PCR. The variation coefficients of intra-assay and inter-assay were both less than 2.5%. The results of clinical application showed that the positive rate was 53.57%,which was obiously better than conventional PCR test, bacterial isolation and identification. This method could be used to detect two diseases simultaneously, and it laid the foundation for subsequent treatment.
引文
[1]李静怡,张建民,徐成刚,等.副猪嗜血杆菌主要毒力因子和致病机理的研究进展[J].中国畜牧兽医,2010, 37(12):173-176.
[2]司振书,王桂英.副猪嗜血杆菌病研究进展[J].中国畜牧兽医,2011,38(06):179-182.
[3] Zhang Bin, Tang Chen, Liao Ming, et al. Update on the pathogenesis of Haemophilus parasuis infection and virulence factors[J]. Vet Microbiol,2014, 168(1):1-7.
[4] Cardoso-Toset F,Gomez-Laguna J,Callejo M, et al. Septicaemic pasteurellosis in free-range pigs associated with an unusual biovar 13 of Pasteurella multocida[J]. Vet Microbiol,2013, 167(3-4):690-694.
[5]梁乔,杨本,勇石霖,等.副猪嗜血杆菌PCR检测方法的建立及应用[J].现代畜牧兽医,2018,358(09):10-13.
[6]张媛,李建,李伟杰,等.PCR检测方法在多杀性巴氏杆菌定种中的应用[J].中国兽药杂志,2017, 51(05):16-21.
[7] Kim T G, Knudsen G R. Comparison of real-time PCR and microscopy to evaluate sclerotial colonisation by a biocontrol fungus[J]. Fungal Biol, 2011, 115(4-5):317-325.
[8]龚雪蕊,李阿茜,刘洋,等.寨卡病毒、登革病毒、基孔肯雅病毒三重荧光定量RT-PCR检测方法的建立及评价[J].病毒学报,2018,34(01):52-58.
[9]李丹丹,徐义刚,邱索平,等.三种鱼类链球菌三重实时荧光PCR方法的建立[J].食品工业,2016,37(09):268-272.
[10]林碧莲,柯振华,陈筱婷,等.多重实时荧光定量PCR快速检测婴幼儿奶粉中沙门氏菌、克罗诺杆菌和金黄色葡萄球菌[J].食品安全质量检测学报,2017,8(11):4375-4381.
[11] Hu Qing-hua, Lyu D Y, Shi Xiao-lu, et al. A modified molecular beacons-based multiplex real-time PCR assay for simultaneous detection of eight foodborne pathogens in a single reaction and its application[J]. Foodborne Pathog Dis,2014,11(3):207-214.
[12] Park J Y, Jeon S, Kim J Y, et al. Multiplex real-time polymerase chain reaction assays for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus[J]. Osong Public Health Res Perspect, 2013, 4(3):133-139.
[13]何世成,周其伟,张浩旭,等.同时检测CSFV、PRRSV、PRV及PCV2多重荧光定量PCR方法的建立[J].中国预防兽医学报,2015,37(11):850-855.
[14] Xie Zhi-xun, Luo Si-si, Xie Li-ji, et al. Simultaneous typing of nine avian respiratory pathogens using a novel GeXP analyzerbased multiplex PCR assay[J]. J Virol Methods, 2014, 207(10):188-195.
[15]薛国聪,任涛.副猪嗜血杆菌病流行病学及致病因子的研究进展[J].中国畜牧兽医,2009,36(05):168-171.
[16] Harper M,Boyce J D.The Myriad Properties of Pasteurella multocida Lipopolysaccharide[J]. Toxins, 2017, 21, 9(8):254-275.
[17]李军,谢宇舟,榻雄标,等.副猪嗜血杆菌实时荧光PCR快速检测方法的建立和应用[J].安徽农业科学,2011,39(06):3607-3609.
[18]姜文灿,岳素文,江洪,等.TaqMan探针法实时荧光定量PCR的应用和研究进展[J].临床检验杂志(电子版),2015, 4(01):797-805.
[19] Huangfu He-ping, Xu Wen-bo, Wang Hong-kui, et al. Detection of Gallibacterium anatis by TaqMan fluorescent quantitative PCR[J]. Avian Pathol, 2018, 47(3):245-252.
[20]陈明玉,孙乐栋,赵佳,等.系统性红斑狼疮合并深部真菌感染多重荧光定量PCR检测研究[J].南方医科大学学报,2009, 29(10):2112-2114.
[21]陈申秒,李郁,王金生,等.副猪嗜血杆菌、多杀性巴氏杆菌和胸膜肺炎放线杆菌多重PCR检测方法的建立及应用[J].畜牧与兽医,2010,42(11):7-10.
[22]朱新贵,田亚平,王亚珊,等.猪多杀性巴氏杆菌和副猪嗜血杆菌双重及多重PCR检测方法的建立[J].黑龙江畜牧兽医,2018,(08):75-78.
[2]司振书,王桂英.副猪嗜血杆菌病研究进展[J].中国畜牧兽医,2011,38(06):179-182.
[3] Zhang Bin, Tang Chen, Liao Ming, et al. Update on the pathogenesis of Haemophilus parasuis infection and virulence factors[J]. Vet Microbiol,2014, 168(1):1-7.
[4] Cardoso-Toset F,Gomez-Laguna J,Callejo M, et al. Septicaemic pasteurellosis in free-range pigs associated with an unusual biovar 13 of Pasteurella multocida[J]. Vet Microbiol,2013, 167(3-4):690-694.
[5]梁乔,杨本,勇石霖,等.副猪嗜血杆菌PCR检测方法的建立及应用[J].现代畜牧兽医,2018,358(09):10-13.
[6]张媛,李建,李伟杰,等.PCR检测方法在多杀性巴氏杆菌定种中的应用[J].中国兽药杂志,2017, 51(05):16-21.
[7] Kim T G, Knudsen G R. Comparison of real-time PCR and microscopy to evaluate sclerotial colonisation by a biocontrol fungus[J]. Fungal Biol, 2011, 115(4-5):317-325.
[8]龚雪蕊,李阿茜,刘洋,等.寨卡病毒、登革病毒、基孔肯雅病毒三重荧光定量RT-PCR检测方法的建立及评价[J].病毒学报,2018,34(01):52-58.
[9]李丹丹,徐义刚,邱索平,等.三种鱼类链球菌三重实时荧光PCR方法的建立[J].食品工业,2016,37(09):268-272.
[10]林碧莲,柯振华,陈筱婷,等.多重实时荧光定量PCR快速检测婴幼儿奶粉中沙门氏菌、克罗诺杆菌和金黄色葡萄球菌[J].食品安全质量检测学报,2017,8(11):4375-4381.
[11] Hu Qing-hua, Lyu D Y, Shi Xiao-lu, et al. A modified molecular beacons-based multiplex real-time PCR assay for simultaneous detection of eight foodborne pathogens in a single reaction and its application[J]. Foodborne Pathog Dis,2014,11(3):207-214.
[12] Park J Y, Jeon S, Kim J Y, et al. Multiplex real-time polymerase chain reaction assays for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus[J]. Osong Public Health Res Perspect, 2013, 4(3):133-139.
[13]何世成,周其伟,张浩旭,等.同时检测CSFV、PRRSV、PRV及PCV2多重荧光定量PCR方法的建立[J].中国预防兽医学报,2015,37(11):850-855.
[14] Xie Zhi-xun, Luo Si-si, Xie Li-ji, et al. Simultaneous typing of nine avian respiratory pathogens using a novel GeXP analyzerbased multiplex PCR assay[J]. J Virol Methods, 2014, 207(10):188-195.
[15]薛国聪,任涛.副猪嗜血杆菌病流行病学及致病因子的研究进展[J].中国畜牧兽医,2009,36(05):168-171.
[16] Harper M,Boyce J D.The Myriad Properties of Pasteurella multocida Lipopolysaccharide[J]. Toxins, 2017, 21, 9(8):254-275.
[17]李军,谢宇舟,榻雄标,等.副猪嗜血杆菌实时荧光PCR快速检测方法的建立和应用[J].安徽农业科学,2011,39(06):3607-3609.
[18]姜文灿,岳素文,江洪,等.TaqMan探针法实时荧光定量PCR的应用和研究进展[J].临床检验杂志(电子版),2015, 4(01):797-805.
[19] Huangfu He-ping, Xu Wen-bo, Wang Hong-kui, et al. Detection of Gallibacterium anatis by TaqMan fluorescent quantitative PCR[J]. Avian Pathol, 2018, 47(3):245-252.
[20]陈明玉,孙乐栋,赵佳,等.系统性红斑狼疮合并深部真菌感染多重荧光定量PCR检测研究[J].南方医科大学学报,2009, 29(10):2112-2114.
[21]陈申秒,李郁,王金生,等.副猪嗜血杆菌、多杀性巴氏杆菌和胸膜肺炎放线杆菌多重PCR检测方法的建立及应用[J].畜牧与兽医,2010,42(11):7-10.
[22]朱新贵,田亚平,王亚珊,等.猪多杀性巴氏杆菌和副猪嗜血杆菌双重及多重PCR检测方法的建立[J].黑龙江畜牧兽医,2018,(08):75-78.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |