绵羊肺炎支原体环介导等温扩增联合横向流动试纸条可视化检测方法的建立
|
摘要
为建立绵羊肺炎支原体快速检测方法,用环介导等温扩增(LAMP)技术进行核酸扩增,通过横向流动试纸条(LFD)实现可视化检测。针对绵羊肺炎支原体HSP 70基因设计一套特异性引物,其中HSP70FIP由生物素标记进行LAMP扩增反应,产物与生物素标记的探针杂交后,在LFD上完成检测,建立了绵羊肺炎支原体LAMP-LFD快速检测方法,并对其特异性、敏感性及临床应用进行检测。结果表明,LAMP在62℃反应70min,即可特异性的检测绵羊肺炎支原体的存在,最低检测线为1.0×102 CFU/mL,灵敏度是常规PCR检测方法的100倍,且对已知感染绵羊肺炎支原体的羊病变肺组织临床样品的检出率为100%。建立的LAMP-LFD检测绵羊肺炎支原体的方法特异性强、灵敏度高并且操作简便,为羊感染绵羊肺炎支原体的预防和诊断提供了新方法。
In order to establish a rapid detection method for Mycoplasma ovipneumoniae,this study used the loop-mediated isothermal amplification(LAMP)technique to carry out nucleic acid amplification and chromatographic visualization via a lateral flow dipstick(LFD)assay.The M.ovipneumoniae heat shock protein70 gene(HSP 70)was detected using a set of specific primers designed for the HSP 70 gene,and the HSP70 FIP was detected by biotin labeling,which was used in the LAMP amplification reaction.The digoxin-labeled probe specifically hybridized with LAMP products,which were visually detected by LFD.Here,we established the M.ovipneumoniae LAMP-LFD rapid detection method and tested the specificity,sensitivity,and clinical application of this method.Results showed that the optimized LAMP performed at 62℃for70 min,and LFD can specifically and visually detect M.ovipneumoniae with a minimum detectable concentration at 1.0×102 CFU/mL.The sensitivity of LAMP-LFD was 102 times than that of the conventional PCR detection methods,and the clinical lung tissue detection rate was 100% of 20 sheep infected with M.ovipneumoniae.In conclusion,LAMP-LFD was established in this study to detect M.ovipneumoniae,a method that was highly specific,sensitive,and easy to operate,and provided a new method for the prevention and diagnosis of M.ovipneumoniaeinfection.
In order to establish a rapid detection method for Mycoplasma ovipneumoniae,this study used the loop-mediated isothermal amplification(LAMP)technique to carry out nucleic acid amplification and chromatographic visualization via a lateral flow dipstick(LFD)assay.The M.ovipneumoniae heat shock protein70 gene(HSP 70)was detected using a set of specific primers designed for the HSP 70 gene,and the HSP70 FIP was detected by biotin labeling,which was used in the LAMP amplification reaction.The digoxin-labeled probe specifically hybridized with LAMP products,which were visually detected by LFD.Here,we established the M.ovipneumoniae LAMP-LFD rapid detection method and tested the specificity,sensitivity,and clinical application of this method.Results showed that the optimized LAMP performed at 62℃for70 min,and LFD can specifically and visually detect M.ovipneumoniae with a minimum detectable concentration at 1.0×102 CFU/mL.The sensitivity of LAMP-LFD was 102 times than that of the conventional PCR detection methods,and the clinical lung tissue detection rate was 100% of 20 sheep infected with M.ovipneumoniae.In conclusion,LAMP-LFD was established in this study to detect M.ovipneumoniae,a method that was highly specific,sensitive,and easy to operate,and provided a new method for the prevention and diagnosis of M.ovipneumoniaeinfection.
引文
[1]Chen C,Qiao J,Meng Q,et al.Serological and molecular survey of sheep infected with Mycoplasma ovipneumoniae,in Xinjiang,China[J].Tropical Animal Health&Production,2015,47(8):1-7.
[2]Nicholas R A J.Improvements in the diagnosis and control of diseases of small ruminants caused by mycoplasmas[J].Small Ruminant Res,2002,45(2):145-149.
[3]Xin J,Li Y,Nicholas R A J,et al.A history of the prevalence and control of contagious bovine pleuropneumonia in China[J].Vet J,2012,191(2):166-170.
[4]卢海亭,乔军,孟庆玲,等.绵羊肺炎支原体p74基因重组单核细胞增生李斯特菌的构建及其免疫原性分析[J].西北农林科技大学学报:自然科学版,2017,45(8):7-14.
[5]杜智慧,沈文,刘海燕,等.巴什拜羊与其杂交羊对绵羊肺炎支原体感染的比较[J].动物医学进展,2017,38(1):69-73.
[6]Fei J,He J,Navarroalvarez N,et al.Elongation factor Tu and heat shock protein 70are membrane-associated proteins from Mycoplasma ovipneumoniae capable of inducing strong immune response in mice[J].PLoS One,2016,11(8):e0161170.8
[7]Ziegler J C,Lahmers K K,Barrington G M,et al.Safety and immunogenicity of a Mycoplasma ovipneumoniae bacterin for domestic sheep(Ovis aries)[J].PLoS One,2014,9(9):e95698.
[8]杜鲜,乔军,陈诚,等.绵羊肺炎支原体降落PCR-侧向层析快速检测方法的建立[J].中国预防兽医学报,2017,39(6):466-470.
[9]Besser T E,Cassirer E F,Potter K A,et al.Epizootic pneumonia of bighorn sheep following experimental exposure to Mycoplasma ovipneumoniae[J].PLoS One,2014,9(10):e110039.
[10]Domesle K J,Yang Q,Hammack T S,et al.Validation of a Salmonella loop-mediated isothermal amplification assay in animal food[J].Int J Food Microbiol,2018,264:63-76.
[11]别闯南,王权,白雪瑞,等.检测创伤弧菌两种毒力基因的环介导等温扩增检测方法的建立[J].中国动物传染病学报,2018(1):48-54.
[12]Lalle M,Possenti A,Dubey J P,et al.Loop-mediated isothermal amplification-lateral-flow dipstick(LAMP-LFD)to detect Toxoplasma gondii oocyst in ready-to-eat salad[J].Food Microbiol,2018,70:137-142.
[13]蔡怡,周前进,陈炯.环介导等温扩增联合横向侧流试纸(LAMP-LFD)对嗜水气单胞菌快速检测方法的建立[J].中国兽医学报,2016,36(2):256-264.
[14]Jaroenram W,Kiatpathomchai W,Flegel T W.Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick[J].Mol Cell Probe,2009,23(2):65-70.
[15]Kongkasuriyachai D,Yongkiettrakul S,Kiatpathomchai W,et al.Loop-mediated isothermal amplification and LFD combination for detection of Plasmodium falciparumand Plasmodium vivax[J].Methods Mol Biol,2017,1572:431-443.
[16]Bottinelli M,Schnee C,Lepri E,et al.Investigation on mycoplasma populations in pneumonic dairy lamb lungs using a DNA microarray assay[J].Small Ruminant Res,2017,147:96-100.
[17]Besser T E,Cassirer E F,Potter K A,et al.Exposure of bighorn sheep to domestic goats colonized with Mycoplasma ovipneumoniae induces sub-lethal pneumonia.[J].PLoS One,2017,12(6):e0178707.
[18]马媛,管礼麟,刀筱芳,等.绵羊肺炎支原体P109'蛋白的分子特征及抗原性分析[J].畜牧与兽医,2017(6):11-13.
[19]田路路,孟庆玲,乔军,等.绵羊肺炎支原体多表位融合基因MO-meAg1的构建、表达及重组蛋白免疫原性分析[J].西北农业学报,2017,26(5):678-684.
[20]史晓娜,王晓晖,高媛,等.基于转录组测序结果对绵羊肺炎支原体培养基优化的初步研究[J].中国兽医科学,2018(1):62-68.
[21]Yang Y,Li Q,Wang S,et al.Rapid and sensitive detection of Babesia bovis and Babesia bigemina by loop-mediated isothermal amplification combined with a lateral flow dipstick[J].Vet Parasitol,2016,219:71-76.
[22]Deng J,Pei J,Gou H,et al.Rapid and simple detection of Japanese encephalitis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick[J].J Virol Method,2014,213:98-105.
[23]Rigano L A,Malamud F,Orce I G,et al.Rapid and sensitive detection of Candidatus liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick[J].BMC Microbiol,2014,6;14:86.doi:10.1186/1471-2180-14-86.
[24]Sun Y L,Yen C H,Ching-Fu T U.Visual detection of canine parvovirus based on loop-mediated isothermal amplification combined with enzyme-linked immunosorbent assay and with lateral flow dipstick[J].J Vet Med Sci,2014,76(4):509-516.
[25]李秀梅,石瑜,朱雅宁,等.环介导等温扩增技术与横向流动试纸条法快速检测鸡贫血病毒的研究[J].中国动物传染病学报,2018(1):25-31.
[26]乔艳,周前进,李孝军,等.内弯宫脂线虫环介导等温扩增联合横向流动试纸条检测方法的建立[J].中国预防兽医学报,2017,39(2):137-142.
[27]柴方超,周前进,陈炯.环介导等温扩增联合横向流动试纸条可视化检测迟缓爱德华菌[J].中国兽医学报,2017,37(6):1103-1110.
[2]Nicholas R A J.Improvements in the diagnosis and control of diseases of small ruminants caused by mycoplasmas[J].Small Ruminant Res,2002,45(2):145-149.
[3]Xin J,Li Y,Nicholas R A J,et al.A history of the prevalence and control of contagious bovine pleuropneumonia in China[J].Vet J,2012,191(2):166-170.
[4]卢海亭,乔军,孟庆玲,等.绵羊肺炎支原体p74基因重组单核细胞增生李斯特菌的构建及其免疫原性分析[J].西北农林科技大学学报:自然科学版,2017,45(8):7-14.
[5]杜智慧,沈文,刘海燕,等.巴什拜羊与其杂交羊对绵羊肺炎支原体感染的比较[J].动物医学进展,2017,38(1):69-73.
[6]Fei J,He J,Navarroalvarez N,et al.Elongation factor Tu and heat shock protein 70are membrane-associated proteins from Mycoplasma ovipneumoniae capable of inducing strong immune response in mice[J].PLoS One,2016,11(8):e0161170.8
[7]Ziegler J C,Lahmers K K,Barrington G M,et al.Safety and immunogenicity of a Mycoplasma ovipneumoniae bacterin for domestic sheep(Ovis aries)[J].PLoS One,2014,9(9):e95698.
[8]杜鲜,乔军,陈诚,等.绵羊肺炎支原体降落PCR-侧向层析快速检测方法的建立[J].中国预防兽医学报,2017,39(6):466-470.
[9]Besser T E,Cassirer E F,Potter K A,et al.Epizootic pneumonia of bighorn sheep following experimental exposure to Mycoplasma ovipneumoniae[J].PLoS One,2014,9(10):e110039.
[10]Domesle K J,Yang Q,Hammack T S,et al.Validation of a Salmonella loop-mediated isothermal amplification assay in animal food[J].Int J Food Microbiol,2018,264:63-76.
[11]别闯南,王权,白雪瑞,等.检测创伤弧菌两种毒力基因的环介导等温扩增检测方法的建立[J].中国动物传染病学报,2018(1):48-54.
[12]Lalle M,Possenti A,Dubey J P,et al.Loop-mediated isothermal amplification-lateral-flow dipstick(LAMP-LFD)to detect Toxoplasma gondii oocyst in ready-to-eat salad[J].Food Microbiol,2018,70:137-142.
[13]蔡怡,周前进,陈炯.环介导等温扩增联合横向侧流试纸(LAMP-LFD)对嗜水气单胞菌快速检测方法的建立[J].中国兽医学报,2016,36(2):256-264.
[14]Jaroenram W,Kiatpathomchai W,Flegel T W.Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick[J].Mol Cell Probe,2009,23(2):65-70.
[15]Kongkasuriyachai D,Yongkiettrakul S,Kiatpathomchai W,et al.Loop-mediated isothermal amplification and LFD combination for detection of Plasmodium falciparumand Plasmodium vivax[J].Methods Mol Biol,2017,1572:431-443.
[16]Bottinelli M,Schnee C,Lepri E,et al.Investigation on mycoplasma populations in pneumonic dairy lamb lungs using a DNA microarray assay[J].Small Ruminant Res,2017,147:96-100.
[17]Besser T E,Cassirer E F,Potter K A,et al.Exposure of bighorn sheep to domestic goats colonized with Mycoplasma ovipneumoniae induces sub-lethal pneumonia.[J].PLoS One,2017,12(6):e0178707.
[18]马媛,管礼麟,刀筱芳,等.绵羊肺炎支原体P109'蛋白的分子特征及抗原性分析[J].畜牧与兽医,2017(6):11-13.
[19]田路路,孟庆玲,乔军,等.绵羊肺炎支原体多表位融合基因MO-meAg1的构建、表达及重组蛋白免疫原性分析[J].西北农业学报,2017,26(5):678-684.
[20]史晓娜,王晓晖,高媛,等.基于转录组测序结果对绵羊肺炎支原体培养基优化的初步研究[J].中国兽医科学,2018(1):62-68.
[21]Yang Y,Li Q,Wang S,et al.Rapid and sensitive detection of Babesia bovis and Babesia bigemina by loop-mediated isothermal amplification combined with a lateral flow dipstick[J].Vet Parasitol,2016,219:71-76.
[22]Deng J,Pei J,Gou H,et al.Rapid and simple detection of Japanese encephalitis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick[J].J Virol Method,2014,213:98-105.
[23]Rigano L A,Malamud F,Orce I G,et al.Rapid and sensitive detection of Candidatus liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick[J].BMC Microbiol,2014,6;14:86.doi:10.1186/1471-2180-14-86.
[24]Sun Y L,Yen C H,Ching-Fu T U.Visual detection of canine parvovirus based on loop-mediated isothermal amplification combined with enzyme-linked immunosorbent assay and with lateral flow dipstick[J].J Vet Med Sci,2014,76(4):509-516.
[25]李秀梅,石瑜,朱雅宁,等.环介导等温扩增技术与横向流动试纸条法快速检测鸡贫血病毒的研究[J].中国动物传染病学报,2018(1):25-31.
[26]乔艳,周前进,李孝军,等.内弯宫脂线虫环介导等温扩增联合横向流动试纸条检测方法的建立[J].中国预防兽医学报,2017,39(2):137-142.
[27]柴方超,周前进,陈炯.环介导等温扩增联合横向流动试纸条可视化检测迟缓爱德华菌[J].中国兽医学报,2017,37(6):1103-1110.