绵羊肺炎支原体和丝状支原体山羊亚种双重TaqMan探针荧光定量PCR检测方法的建立及应用
|
摘要
绵羊肺炎支原体(Mycoplasma ovipneumoniae, Mo)和丝状支原体山羊亚种(M. mycoides subsp.capri, Mmc)是引起羊支原体性肺炎(M. pneumonia of sheep and goats, MPSG)的主要病原。为了快速鉴别引起MPSG的主要病原,建立一种可以同时检测Mo和Mmc的双重TaqMan探针荧光定量PCR (Realtime fluorescence quantitative PCR, q RT-PCR)检测方法。根据Mo p113序列和Mmc MLC_1770(hypothetical protein gene)序列,利用Beacon Designer 7.9结合NCBI Blast软件分析,分别设计出针对Mo和Mmc的特异性引物和探针。通过对引物浓度、探针浓度和退火温度等反应条件的优化,建立了双重TaqMan探针qRT-PCR方法,并对该方法的特异性、敏感性和重复性进行了验证。结果表明,建立的双重TaqMan探针qRT-PCR的标准曲线相关系数R2分别为0.998和0.999,扩增效率分别为94.8%和97.0%;该方法对其他羊常见病原均无扩增信号,证明该方法具有良好的特异性;该方法对Mo和Mmc的最低检测限均为100 copies/μL,敏感性是常规PCR的100倍;组内和组间变异系数都低于2%,说明重复性好。应用该方法对福建省内收集的187例临床样品进行检测,结果显示,Mo的阳性率为47.6%(89/187),Mmc的阳性率为11.8%(22/187),两种病原体混合感染阳性率为10.2%(19/187)。以上数据结果表明,本研究建立的方法可用于临床上Mo和Mmc的准确、快速检测。本研究为Mo和Mmc的快速检测及流行病学提供了技术支撑。
Mycoplasma ovipneumoniae(Mo) and M. mycoides subsp. capri(Mmc) are the main pathogens of M. pneumonia of sheep and goats(MPGS). In order to rapidly identify the main pathogens of MPGS, a duplex TaqMan probe Real-time fluorescence quantitative PCR(qRT-PCR) detection method was established for simultaneous detection of Mo and Mmc. Using Beacon Designer 7.9 combined with NCBI Blast software analysis, the specific primers and probes were designed based on the p113 sequence of Mo and the MLC_1770(hypothetical protein gene) sequence of Mmc. The duplex TaqMan probe qRT-PCR method was established by optimizing the reaction conditions such as primer concentration, probe concentration and annealing temperature, and the specificity, sensitivity and repeatability of the method were validated. The result showed that the correlation coefficient(R2) of Mo and Mmc were 0.998 and 0.999, respectively, and the amplification efficiency of Mo and Mmc were 94.8% and 97%, respectively. The assay showed a good specificity without cross reaction from other common pathogens of sheep and goats. The lowest detectable limit(LDL) of the method for Mo and Mmc was the same, which was 100 copies/μL, and its sensitivity was 100 times more than conventional PCR. The inter-and intra-variation coefficients of the assay were less than 2%. Applying the assay to detect 187 cases of clinical samples which were collected from different areas of Fujian, the result showed that the positive rates of Mo and Mmc were 47.6%(89/187) and 11.8%(22/187), respectively, and the duplex positive rate of infection was 10.2%(19/187). The above results indicated that the assay could be used for the accurate and rapid detection of Mo and Mmc in clinic. The study provided technical support for rapid detection and epidemiology of Mo and Mmc.
Mycoplasma ovipneumoniae(Mo) and M. mycoides subsp. capri(Mmc) are the main pathogens of M. pneumonia of sheep and goats(MPGS). In order to rapidly identify the main pathogens of MPGS, a duplex TaqMan probe Real-time fluorescence quantitative PCR(qRT-PCR) detection method was established for simultaneous detection of Mo and Mmc. Using Beacon Designer 7.9 combined with NCBI Blast software analysis, the specific primers and probes were designed based on the p113 sequence of Mo and the MLC_1770(hypothetical protein gene) sequence of Mmc. The duplex TaqMan probe qRT-PCR method was established by optimizing the reaction conditions such as primer concentration, probe concentration and annealing temperature, and the specificity, sensitivity and repeatability of the method were validated. The result showed that the correlation coefficient(R2) of Mo and Mmc were 0.998 and 0.999, respectively, and the amplification efficiency of Mo and Mmc were 94.8% and 97%, respectively. The assay showed a good specificity without cross reaction from other common pathogens of sheep and goats. The lowest detectable limit(LDL) of the method for Mo and Mmc was the same, which was 100 copies/μL, and its sensitivity was 100 times more than conventional PCR. The inter-and intra-variation coefficients of the assay were less than 2%. Applying the assay to detect 187 cases of clinical samples which were collected from different areas of Fujian, the result showed that the positive rates of Mo and Mmc were 47.6%(89/187) and 11.8%(22/187), respectively, and the duplex positive rate of infection was 10.2%(19/187). The above results indicated that the assay could be used for the accurate and rapid detection of Mo and Mmc in clinic. The study provided technical support for rapid detection and epidemiology of Mo and Mmc.
引文
程振涛,张双翔,王慧,等.2013.绵羊肺炎支原体实时荧光定量PCR检测方法的建立[J].西北农业学报,22(2):7-12.(Cheng Z T,Zhang S X,Wang H,et al.2013.Establishment of real-time fluorescent quantitative PCR assay for detection of Mycoplasma ovipneumoniae[J].Acta Agriculturae Boreali-occidentalis Sinica,22(2):7-12.)
储岳峰,高鹏程,赵萍,等.2009.应用双重PCR方法检测羊支原体性肺炎病原[J].畜牧与兽医,41(12):23-26.(Chu Y F,Gao P C,Zhao P,et al.2009.Development of a duplex polymerase chain reaction assay for the detection of pathogens for Mycoplasmal pneumonia of sheep and goats[J].Animal Husbandry&Veterinary Medicine,41(12):23-26.)
凤英,高娃,陈伟,等.2015.一株致病性丝状支原体山羊亚种支原体的分离鉴定[J].畜牧与饲料科学,36(2):4-8.(Feng Y,Gao Y,Chen W,et al.2015.Characterization of a pathogenic Mycoplasma mycoides subsp.capri isolate[J].Animal Husbandry and Feed Science,36(2):4-8.)
黄坚,尹正军,岳华,等.2016.绵羊肺炎支原体荧光定量PCR检测方法的建立与应用[J].中国兽医科学,40(10):1270-1276.(Huang J,Yin Z J,Yue H,et al.2016.Development and application of quantitative real-time PCR for the detection of Mycoplasma ovipneumoniae[J].Chinese Veterinary Science,40(10):1270-1276.)
江锦秀,林甦,林裕胜,等.2015.绵羊肺炎支原体FJ01-CL株的分离和鉴定[J].福建农业学报,30(5):430-434.(Jiang J X,Lin S,Lin Y S,et al.2015.Isolation and identification of Mycoplasma ovipneumoniae FJ01-CL[J].Fujian Journal of Agricultural Sciences,30(5):430-434.)
江锦秀,林裕胜,游伟,等.2016.丝状支原体山羊亚种FJ-GT株的分离和鉴定[J].中国农学通报,32(29):11-16.(Jiang J X,Lin Y S,You W,et al.2016.Isolation and identification of Mycoplasma mycoides subsp.capri FJ-GT[J].Chinese Agricultural Science Bulletin,32(29):11-16.)
江锦秀,林裕胜,游伟,等.2017.福建省羊支原体性肺炎分子流行病学研究[J].福建农业学报,32(1):12-16.(Jiang J X,Lin Y S,You W,et al.2017.A molecular epidemiological study on Mycoplasma pneumonia of sheep and goats in Fujian[J].Fujian Journal of Agricultural Sciences,32(1):12-16.)
林裕胜,江锦秀,张靖鹏,等.2017.丝状支原体山羊亚种SYBR Green I qRT-PCR快速检测方法的建立[J].农业生物技术学报,25(11):1895-1902.(Lin Y S,Jiang J X,Zhang J P,et al.2017.Establishment of a SYBR Green IqRT-PCR for rapid detection of Mycoplasma mycoides subsp.capri[J].Journal of Agricultural Biotechnology,25(11):1895-1902.)
林裕胜,江锦秀,张靖鹏,等.2018.绵羊肺炎支原体TaqMan荧光定量PCR检测方法的建立[J].中国预防兽医学报,40(4):316-320.(Lin Y S,Jiang J X,Zhang J P,et al.2018.Establishment of a TaqMan real-time PCR fluorescent quantitative PCR assay for detection of Mycoplasma ovipneunoniae[J].Chinese Journal of Preventive Veterinary Medicine,40(4):316-320.)
沈志强,王金良,郭显坡,等.2011.SYBR GreenⅠ实时荧光定量PCR检测猪细小病毒方法的建立[J].中国兽医学报,31(1):11-15.(Shen Z Q,Wang J L,Guo X P,et al.2011.Development and application of SYBR-GreenⅠreal-time quantitative PCR technique for detecting porcine parvovirus virus[J].Chinese Journal of Veterinary Science,31(1):11-15.)
王华,杨发龙,王永,等.2011.山羊支原体性肺炎流行病学调查[J].中国畜牧兽医,38(1):210-214.(Wang H,Yang F L,Wang Y,et al.2011.Epidemiological investigation of caprine Mycoplasma pneumoniae in Sichuan province[J].China Animal Husbandry&Veterinary Medicine,38(1):210-214.)
吴燕,王琦,周碧君,等.2017.绵羊肺炎支原体贵州流行株P113基因序列分析[J].中国兽医学报,36(5):756-762.(Wu Y,Wang Q,Zhou B J,et al.2017.Sequence analysis of P113 gene of Mycoplasma ovipneumoniae strains epidemic in Guizhou province[J].Chinese Veterinary Science,36(5):756-762.)
向志龙,卓建华,鲜思美,等.2011.羊口疮病毒环介导等温扩增快速检测方法的建立及应用[J].中国兽医科学,41(06):588-592.(Xiang Z L,Zhuo J H,Xian S M,et al.2011.Establishment and application of a loop-mediated isothermal amplification method for rapid detection of orf virus[J].Chinese Veterinary Science,41(06):588-592.)
Amores J,Corrales J C,Gómez Martin A G,et al.2010.Comparison of culture and PCR to detect Mycoplasma agalactiae and Mycoplasma mycoides subsp.capri in ear swabs taken from goats[J].Veterinary microbiology,140(1-2):105-108.
Besser T E,Cassirer E F,Potter K A,et al.2008.Association of Mycoplasma ovipneumoniae infection with populationlimiting respiratory disease in free-ranging Rocky Mountain bighorn sheep(Ovis canadensis canadensis)[J].Journal of Clinical Microbiology,46(2):423-430.
Dassanayke R P,Shanthalingam S,Hemdon C N,et al.2010.Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia[J].Veterinary Microbiology,145(3-4):354-359.
Jones G E,Wood A R.1988.Microbiological and serological studies on caprine pneumonias Oman[J].Research in Veterinary Science,44(1):125-131.
Kibe M K,Bidwell D E,Turp P,et al.1985.Demonstration of cross-reactive antigens in F38 and related mycoplasmas by enzyme-linked immunosorbent assay(ELISA)and immunoblotting[J].The Journal of Hygiene,95(1):95-106.
McAuliffe L,Hatchell F M,Ayling R D,et al.2003.Detection of Mycoplasma ovipneumoniae in Pasteurella-vaccinated sheep flocks with respiratory disease in England[J].Veterinary Record,153(22):687-8.
Rifatbegovic M,Maksimovic Z,Hulaj B,et al.2011.Mycoplasma ovipneumoniae associated with severe respiratory disease in goats[J].Veterinary Record,168(21):565.
Sharew A D,Staak C,Thiaucourt F,et al.2005.A serological investigation into contagious caprine pleuropneumonia(CCPP)in Ethiopia[J].Tropical Animal Health and Production,37(1):11-19.
Thirkell D,Spooner R K,Jones G E,et al.1991.Cross-reacting antigens between Mycoplasma ovipneumoniae and other species of mycoplasma of animal orgin,shown by ELISA and immunoblotting with reference antisera[J].Veterinary Microbiology,26(3):249-261.
Yang F,Dao X,Rodriguez-Palacios A,et al.2014.A real-time PCR for detection and quantification of Mycoplasma ovipneumoniae[J].Journal of Veterinary Medical Science,76(12):1631-1634.
储岳峰,高鹏程,赵萍,等.2009.应用双重PCR方法检测羊支原体性肺炎病原[J].畜牧与兽医,41(12):23-26.(Chu Y F,Gao P C,Zhao P,et al.2009.Development of a duplex polymerase chain reaction assay for the detection of pathogens for Mycoplasmal pneumonia of sheep and goats[J].Animal Husbandry&Veterinary Medicine,41(12):23-26.)
凤英,高娃,陈伟,等.2015.一株致病性丝状支原体山羊亚种支原体的分离鉴定[J].畜牧与饲料科学,36(2):4-8.(Feng Y,Gao Y,Chen W,et al.2015.Characterization of a pathogenic Mycoplasma mycoides subsp.capri isolate[J].Animal Husbandry and Feed Science,36(2):4-8.)
黄坚,尹正军,岳华,等.2016.绵羊肺炎支原体荧光定量PCR检测方法的建立与应用[J].中国兽医科学,40(10):1270-1276.(Huang J,Yin Z J,Yue H,et al.2016.Development and application of quantitative real-time PCR for the detection of Mycoplasma ovipneumoniae[J].Chinese Veterinary Science,40(10):1270-1276.)
江锦秀,林甦,林裕胜,等.2015.绵羊肺炎支原体FJ01-CL株的分离和鉴定[J].福建农业学报,30(5):430-434.(Jiang J X,Lin S,Lin Y S,et al.2015.Isolation and identification of Mycoplasma ovipneumoniae FJ01-CL[J].Fujian Journal of Agricultural Sciences,30(5):430-434.)
江锦秀,林裕胜,游伟,等.2016.丝状支原体山羊亚种FJ-GT株的分离和鉴定[J].中国农学通报,32(29):11-16.(Jiang J X,Lin Y S,You W,et al.2016.Isolation and identification of Mycoplasma mycoides subsp.capri FJ-GT[J].Chinese Agricultural Science Bulletin,32(29):11-16.)
江锦秀,林裕胜,游伟,等.2017.福建省羊支原体性肺炎分子流行病学研究[J].福建农业学报,32(1):12-16.(Jiang J X,Lin Y S,You W,et al.2017.A molecular epidemiological study on Mycoplasma pneumonia of sheep and goats in Fujian[J].Fujian Journal of Agricultural Sciences,32(1):12-16.)
林裕胜,江锦秀,张靖鹏,等.2017.丝状支原体山羊亚种SYBR Green I qRT-PCR快速检测方法的建立[J].农业生物技术学报,25(11):1895-1902.(Lin Y S,Jiang J X,Zhang J P,et al.2017.Establishment of a SYBR Green IqRT-PCR for rapid detection of Mycoplasma mycoides subsp.capri[J].Journal of Agricultural Biotechnology,25(11):1895-1902.)
林裕胜,江锦秀,张靖鹏,等.2018.绵羊肺炎支原体TaqMan荧光定量PCR检测方法的建立[J].中国预防兽医学报,40(4):316-320.(Lin Y S,Jiang J X,Zhang J P,et al.2018.Establishment of a TaqMan real-time PCR fluorescent quantitative PCR assay for detection of Mycoplasma ovipneunoniae[J].Chinese Journal of Preventive Veterinary Medicine,40(4):316-320.)
沈志强,王金良,郭显坡,等.2011.SYBR GreenⅠ实时荧光定量PCR检测猪细小病毒方法的建立[J].中国兽医学报,31(1):11-15.(Shen Z Q,Wang J L,Guo X P,et al.2011.Development and application of SYBR-GreenⅠreal-time quantitative PCR technique for detecting porcine parvovirus virus[J].Chinese Journal of Veterinary Science,31(1):11-15.)
王华,杨发龙,王永,等.2011.山羊支原体性肺炎流行病学调查[J].中国畜牧兽医,38(1):210-214.(Wang H,Yang F L,Wang Y,et al.2011.Epidemiological investigation of caprine Mycoplasma pneumoniae in Sichuan province[J].China Animal Husbandry&Veterinary Medicine,38(1):210-214.)
吴燕,王琦,周碧君,等.2017.绵羊肺炎支原体贵州流行株P113基因序列分析[J].中国兽医学报,36(5):756-762.(Wu Y,Wang Q,Zhou B J,et al.2017.Sequence analysis of P113 gene of Mycoplasma ovipneumoniae strains epidemic in Guizhou province[J].Chinese Veterinary Science,36(5):756-762.)
向志龙,卓建华,鲜思美,等.2011.羊口疮病毒环介导等温扩增快速检测方法的建立及应用[J].中国兽医科学,41(06):588-592.(Xiang Z L,Zhuo J H,Xian S M,et al.2011.Establishment and application of a loop-mediated isothermal amplification method for rapid detection of orf virus[J].Chinese Veterinary Science,41(06):588-592.)
Amores J,Corrales J C,Gómez Martin A G,et al.2010.Comparison of culture and PCR to detect Mycoplasma agalactiae and Mycoplasma mycoides subsp.capri in ear swabs taken from goats[J].Veterinary microbiology,140(1-2):105-108.
Besser T E,Cassirer E F,Potter K A,et al.2008.Association of Mycoplasma ovipneumoniae infection with populationlimiting respiratory disease in free-ranging Rocky Mountain bighorn sheep(Ovis canadensis canadensis)[J].Journal of Clinical Microbiology,46(2):423-430.
Dassanayke R P,Shanthalingam S,Hemdon C N,et al.2010.Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia[J].Veterinary Microbiology,145(3-4):354-359.
Jones G E,Wood A R.1988.Microbiological and serological studies on caprine pneumonias Oman[J].Research in Veterinary Science,44(1):125-131.
Kibe M K,Bidwell D E,Turp P,et al.1985.Demonstration of cross-reactive antigens in F38 and related mycoplasmas by enzyme-linked immunosorbent assay(ELISA)and immunoblotting[J].The Journal of Hygiene,95(1):95-106.
McAuliffe L,Hatchell F M,Ayling R D,et al.2003.Detection of Mycoplasma ovipneumoniae in Pasteurella-vaccinated sheep flocks with respiratory disease in England[J].Veterinary Record,153(22):687-8.
Rifatbegovic M,Maksimovic Z,Hulaj B,et al.2011.Mycoplasma ovipneumoniae associated with severe respiratory disease in goats[J].Veterinary Record,168(21):565.
Sharew A D,Staak C,Thiaucourt F,et al.2005.A serological investigation into contagious caprine pleuropneumonia(CCPP)in Ethiopia[J].Tropical Animal Health and Production,37(1):11-19.
Thirkell D,Spooner R K,Jones G E,et al.1991.Cross-reacting antigens between Mycoplasma ovipneumoniae and other species of mycoplasma of animal orgin,shown by ELISA and immunoblotting with reference antisera[J].Veterinary Microbiology,26(3):249-261.
Yang F,Dao X,Rodriguez-Palacios A,et al.2014.A real-time PCR for detection and quantification of Mycoplasma ovipneumoniae[J].Journal of Veterinary Medical Science,76(12):1631-1634.