绵羊肺炎支原体恒温热隔绝式PCR方法的建立
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摘要
为建立绵羊肺炎支原体(Mo)感染疾病的现场便捷化诊断方法,本研究选择Mo高度保守区域tuf基因设计合成一对特异性引物和探针,建立了恒温热隔绝式PCR(iiPCR)方法并对其特异性、灵敏性进行评价。结果显示,建立的ii PCR方法仅对Mo的典型菌株和其临床分离菌株的DNA呈阳性结果,具有较强的特异性;对Mo基因组DNA的检出下限为24 fg/μL,具有较高的敏感性。利用该方法检测临床样品,结果显示该方法可以从临床采集的羊鼻拭子及支气管拭子中检测出Mo DNA,对扩增产物的测序表明检测结果具有较高的准确性。本研究建立的iiPCR方法为Mo的检测及其感染疾病的诊断提供了快速、便捷的技术手段。
In order to develope an on-site and convenient diagnostic method for Mycoplasma ovipneumoniae infection, this study established an insulated isothermal PCR(iiPCR) by using a pair of specific primers and probe targeting the highly conserved region of the M.ovipneumoniae tuf gene. The specificity and sensitivity of the assay were evaluated, and the results showed that the established iiP CR had high specificity for the bacteria detection, as it gave positive results only for the DNA of the M.ovipneumoniae type strains and the clinical isolates of M.ovipneumoniae. The detection limit for the genomic DNA was proven to be 48fg. The feasibility and accuracy of the method to detect clinical samples were also verified. The results demonstrated that the iiP CR was able to detect M.ovipneumoniae from clinically collected nasal swabs and bronchial swabs. Sequencing of the PCR products confirmed the accuracy of the assay. The iiP CR developed in this study provided a rapid and convenient tool for M.ovipneumoniae identification and diagnosis of the virus infected animals.
In order to develope an on-site and convenient diagnostic method for Mycoplasma ovipneumoniae infection, this study established an insulated isothermal PCR(iiPCR) by using a pair of specific primers and probe targeting the highly conserved region of the M.ovipneumoniae tuf gene. The specificity and sensitivity of the assay were evaluated, and the results showed that the established iiP CR had high specificity for the bacteria detection, as it gave positive results only for the DNA of the M.ovipneumoniae type strains and the clinical isolates of M.ovipneumoniae. The detection limit for the genomic DNA was proven to be 48fg. The feasibility and accuracy of the method to detect clinical samples were also verified. The results demonstrated that the iiP CR was able to detect M.ovipneumoniae from clinically collected nasal swabs and bronchial swabs. Sequencing of the PCR products confirmed the accuracy of the assay. The iiP CR developed in this study provided a rapid and convenient tool for M.ovipneumoniae identification and diagnosis of the virus infected animals.
引文
[1]Besser T E,Cassirer E F,Potter K A,et al.Asso ciation of Mycoplasma ovipneumoniae infection with population-limiting respiratory disease in free-ranging rocky mountain bighorn sheep(Ovis canadensis canadensis)[J].J Clin Microbiol,2008,46(2):423-430.
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[4]冯旭飞,张贤宇,王成龙,等.基于p113基因的绵羊肺炎支原体PCR检测方法的建立及应用[J].中国兽医科学,2013,43(11):1157-1161.
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[7]Tsai Y L,Wang H T,Chang H F,et al.Develop ment of TaqMan probe-based insulated isothermal PCR(iiPCR)for sensitive and specific on-site pathogen detec tion[J].PLoS One,2012,7(9):e45278.
[8]Chang H F,Tsai Y L,Tsai C F,et al.A thermally baffled device for highly stabilized convective PCR[J].Biotechnol J,2012,7(5):662-666.
[9]Krishnan M,Ugaz V M,Burns M A.PCR in a Rayleigh-Benard convection cell[J].Science,2002,298(5594):793.
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[11]Lung O,Pasick J,Fisher M,et al.Insulated isothermal reverse transcriptase PCR(iiRT-PCR)for rapid and sensitive detection of classical swine fever virus[J].Transbound Emerg Dis,2015,63(5):395-402.
[12]Balasuriya U B,Lee P Y,Tiwari A,et al.Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR(iiRT-PCR)assay using the POCKITTMNucleic Acid Analyzer[J].J Virol Methods,2014,207:66-72.
[13]Kuo H C,Lo D Y,Chen C L,et al.Rapid and sensitive detection of Mycoplasma synoviae by an insu lated isothermal polymerase chain reaction-based assay on a field-deployable device[J].Poult Sci,2017,96(1):35-41.
[14]Besser T E,Cassirer E F,Potter KA,et al.Exposure of bighorn sheep to domestic goats colonized with Mycoplasma ovipneumoniae induces sub-lethal pneumonia[J].PLoS One,2017,12(6):e0178707.
[15]Rong Guang,Zhao Jun-Ming,Hou Guan-yu,et al.Seroprevalence and molecular detection of Mycoplasma ovipneumoniae in goats in tropical China[J].Trop Anim Health Prod,2014,46(8):1491-1495.
[16]Giangaspero M,Nicholas RA,Hlusek M,et al.Seroepidemiological survey of sheep flocks from Northern Japan for Mycoplasma ovipneumoniae and Mycoplasma agalactiae[J].Trop Anim Health Prod,2012,44(3):395-398.
[17]谢秀兰,额尔和花,黎帅,等.宁夏不同绵羊品种中绵羊肺炎支原体的调查[J].中国兽医杂志,2017,(6):11-13.
[18]冯旭飞,王远微,李定霏,等.绵羊肺炎支原体和溶血性曼氏杆菌双重PCR检测方法的建立及应用[J].中国预防兽医学报,2014,36(10):788-791.
[19]Tsai Y L,Wang H T,Chang H F,et al.Development of TaqMan probe-based insulated isothermal PCR(iiPCR)for sensitive and specific on-site pathogen detection[J].PLo S One,2012,7(9):e45278.
[20]Yang Fa-long,Tang Cheng,Wang Yong,et al.Genome sequence of Mycoplasma ovipneumoniae strain SC01[J].J Bacteriol,2011,193(18):5018.
[2]Dassanayake R P,Shanthalingam S,Herndon C N,et al.Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia[J].Vet Microbiol,2010,145(3-4):354-359.
[3]McA uliffe L,Hatchell F,Ayling R,et al.Detection of Mycoplasma ovipneumoniae in Pasteurella-vaccinated sheep flocks with respiratory disease in England[J].Vet Rec,2003,153:687-688.
[4]冯旭飞,张贤宇,王成龙,等.基于p113基因的绵羊肺炎支原体PCR检测方法的建立及应用[J].中国兽医科学,2013,43(11):1157-1161.
[5]Yang Fa-long,Han Xiao,Tang Cheng,et al.A hsp70 genebased PCR for detection of Mycoplasma ovipneumoniae[J].JAnim Vet Adv,2013,12(19):1495-1497.
[6]Yang Fa-long,Dao Xiao-fang,Rodriguezpalacios A,et al.A real-time PCR for detection and quantifica tion of Mycoplasma ovipneumoniae[J].J Vet Med Sci,2014,(11):1631-1634.
[7]Tsai Y L,Wang H T,Chang H F,et al.Develop ment of TaqMan probe-based insulated isothermal PCR(iiPCR)for sensitive and specific on-site pathogen detec tion[J].PLoS One,2012,7(9):e45278.
[8]Chang H F,Tsai Y L,Tsai C F,et al.A thermally baffled device for highly stabilized convective PCR[J].Biotechnol J,2012,7(5):662-666.
[9]Krishnan M,Ugaz V M,Burns M A.PCR in a Rayleigh-Benard convection cell[J].Science,2002,298(5594):793.
[10]Wilkes R P,Tsai Y L,Lee P Y,et al.Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction[J].BMCVet Res,2014,10(1):1-8.
[11]Lung O,Pasick J,Fisher M,et al.Insulated isothermal reverse transcriptase PCR(iiRT-PCR)for rapid and sensitive detection of classical swine fever virus[J].Transbound Emerg Dis,2015,63(5):395-402.
[12]Balasuriya U B,Lee P Y,Tiwari A,et al.Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR(iiRT-PCR)assay using the POCKITTMNucleic Acid Analyzer[J].J Virol Methods,2014,207:66-72.
[13]Kuo H C,Lo D Y,Chen C L,et al.Rapid and sensitive detection of Mycoplasma synoviae by an insu lated isothermal polymerase chain reaction-based assay on a field-deployable device[J].Poult Sci,2017,96(1):35-41.
[14]Besser T E,Cassirer E F,Potter KA,et al.Exposure of bighorn sheep to domestic goats colonized with Mycoplasma ovipneumoniae induces sub-lethal pneumonia[J].PLoS One,2017,12(6):e0178707.
[15]Rong Guang,Zhao Jun-Ming,Hou Guan-yu,et al.Seroprevalence and molecular detection of Mycoplasma ovipneumoniae in goats in tropical China[J].Trop Anim Health Prod,2014,46(8):1491-1495.
[16]Giangaspero M,Nicholas RA,Hlusek M,et al.Seroepidemiological survey of sheep flocks from Northern Japan for Mycoplasma ovipneumoniae and Mycoplasma agalactiae[J].Trop Anim Health Prod,2012,44(3):395-398.
[17]谢秀兰,额尔和花,黎帅,等.宁夏不同绵羊品种中绵羊肺炎支原体的调查[J].中国兽医杂志,2017,(6):11-13.
[18]冯旭飞,王远微,李定霏,等.绵羊肺炎支原体和溶血性曼氏杆菌双重PCR检测方法的建立及应用[J].中国预防兽医学报,2014,36(10):788-791.
[19]Tsai Y L,Wang H T,Chang H F,et al.Development of TaqMan probe-based insulated isothermal PCR(iiPCR)for sensitive and specific on-site pathogen detection[J].PLo S One,2012,7(9):e45278.
[20]Yang Fa-long,Tang Cheng,Wang Yong,et al.Genome sequence of Mycoplasma ovipneumoniae strain SC01[J].J Bacteriol,2011,193(18):5018.