TaqMan实时荧光定量PCR检测艰难梭菌
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摘要
目的建立特异敏感的实时荧光定量PCR方法,用于艰难梭菌的快速检测;评价基于纯培养艰难梭菌和粪便中艰难梭菌的2种标准曲线;并应用该荧光定量PCR法对急性艰难梭菌感染(CDI)的小鼠进行粪便含菌量检测评价。方法针对艰难梭菌基因组中16S r RNA序列设计特异性引物和探针,建立一套快速检测艰难梭菌含量的实时荧光定量PCR方法,验证方法的特异性、灵敏性;绘制艰难梭菌纯菌浓度梯度稀释标准曲线和粪便中同浓度梯度艰难梭菌的标准曲线,比较两者的差异;用艰难梭菌高毒株NAP1/027感染用抗生素处理的C57BL/6小鼠,建立CDI小鼠模型,同时应用该荧光定量PCR和活菌培养法定量检测小鼠粪便中的艰难梭菌含量变化。结果建立的Taq Man实时荧光定量PCR具有较高的灵敏性和特异性,生成标准曲线的相关系数为0.999 8,斜率为-3.400 4;用纯培养艰难梭菌和粪便中艰难梭菌分别制备标准曲线,结果表明2种标准曲线定量检测结果差异无统计学意义。建立CDI小鼠模型,应用该荧光PCR能有效、准确的检测出粪便中艰难梭菌的含量,可替代费时费力的活菌培养计数法。结论用纯培养艰难梭菌来制备标准曲线不影响对含菌粪便标本的准确定量检测,荧光定量PCR能准确快捷地检测CDI小鼠粪便中的艰难梭菌含量,比活菌计数更快速和方便,可用于艰难梭菌感染小鼠模型中小鼠肠道内艰难梭菌定植的定量检测。
Objective To establish the specific and sensitive real time fluorescence quantitative PCR(RT-PCR)assay for therapid detection of Clostridium difficile,evaluate two standard curves of quantitative using pure bacterial culture or sameconcentration of bacteria in feces and quantitatively measure C. difficile counts in feces of mice infected with C. difficile.Methods The specific primers and probe for Taqman based PCR were designed based on 16 S r RNA sequence of C. difficile. ATaq Man probe based RT-PCR assay was established,and its specificity,sensitivity and standard curve were assessed. The standardcurve based on pure bacterial gradient dilution or the same concentration gradient bacteria in feces were compared to determine theoptimal quantitative RT-PCR. Antibiotic-treated C57 BL/6 mice were infected with virulent C. difficile strain NAP1/027 to establishC. difficile infected(CDI)mouse model. The amounts of C. difficile in fecal samples of CDI mice were detected by using quantitativeRT-PCR and culture method.Results The specificity and sensitivity of this established Taq Man probe based RT-PCR were high.The correlation coefficient and slope value of standard curve were 0.999 8 and-3.400 4 respectively. There was no significantdifference in counts of C. difficile in artificial prepared mimic fecal samples using two standard curves of quantitative RT-PCR basedon pure bacterial gradient dilution or the same concentration gradient bacteria in feces. Antibiotic cocktail pretreated CDI mousemodel was established. The results showed that the quantitative RT-PCR can detect the amount of C. difficile in mice feces with highefficiency and accuracy and can replace the culture method.Conclusion The quantitative RT-PCR is reliable and efficient for thedetection of colonization of C. difficile in CDI mouse model by using standard curve based on pure bacteria compared with theculture method. This RT-PCR assay can be used to detect the C. difficile in CDI mouse model.
Objective To establish the specific and sensitive real time fluorescence quantitative PCR(RT-PCR)assay for therapid detection of Clostridium difficile,evaluate two standard curves of quantitative using pure bacterial culture or sameconcentration of bacteria in feces and quantitatively measure C. difficile counts in feces of mice infected with C. difficile.Methods The specific primers and probe for Taqman based PCR were designed based on 16 S r RNA sequence of C. difficile. ATaq Man probe based RT-PCR assay was established,and its specificity,sensitivity and standard curve were assessed. The standardcurve based on pure bacterial gradient dilution or the same concentration gradient bacteria in feces were compared to determine theoptimal quantitative RT-PCR. Antibiotic-treated C57 BL/6 mice were infected with virulent C. difficile strain NAP1/027 to establishC. difficile infected(CDI)mouse model. The amounts of C. difficile in fecal samples of CDI mice were detected by using quantitativeRT-PCR and culture method.Results The specificity and sensitivity of this established Taq Man probe based RT-PCR were high.The correlation coefficient and slope value of standard curve were 0.999 8 and-3.400 4 respectively. There was no significantdifference in counts of C. difficile in artificial prepared mimic fecal samples using two standard curves of quantitative RT-PCR basedon pure bacterial gradient dilution or the same concentration gradient bacteria in feces. Antibiotic cocktail pretreated CDI mousemodel was established. The results showed that the quantitative RT-PCR can detect the amount of C. difficile in mice feces with highefficiency and accuracy and can replace the culture method.Conclusion The quantitative RT-PCR is reliable and efficient for thedetection of colonization of C. difficile in CDI mouse model by using standard curve based on pure bacteria compared with theculture method. This RT-PCR assay can be used to detect the C. difficile in CDI mouse model.
引文
[1]Sun XM,Hirota SA.The roles of host and pathogen factors and the innate immune response in the pathogenesis of Clostridium difficile infection[J].Mol Immunol,2015,63(2):193-202.DOI:10.1016/j.molimm.2014.09.005.
[2]Kubota H,Sakai T,Gawad A,et al.Development of Taq Manbased quantitative PCR for sensitive and selective detection of toxigenic Clostridium difficile in human stools[J].PLo S One,2014,9(10):e111684.DOI:10.1371/journal.pone.0111684.
[3]Sloan LM,Duresko BJ,Gustafson DR,et al.Comparison of realtime PCR for detection of thet cd C gene with four toxin immunoassays and culture in diagnosis of Clostridium difficile infection[J].J Clin Microbiol,2008,46(6):1996-2001.DOI:10.1128/JCM.00032-08.
[4]Best EL,Freeman J,Wilcox MH.Models for the study of Clostridium difficile infection[J].Gut Microbes,2012,3(2):145-167.DOI:10.4161/gmic.19526.
[5]Bélanger SD,Boissinot M,Clairoux N,et al.Rapid detection of Clostridium difficile in feces by real-time PCR[J].J Clin Microbiol,2003,41(2):730-734.DOI:10.1128/JCM.41.2.730-734.2003.
[6]Chen XH,Katchar K,Goldsmith JD,et al.A mouse model of Clostridium difficile-associated disease[J].Gastroenterology,2008,135(6):1984-1992.DOI:10.1053/j.gastro.2008.09.002.
[7]Britton RA,Young VB.Interaction between the intestinal microbiota and host in Clostridium difficile colonization resistance[J].Trends Microbiol,2012,20(7):313-319.DOI:10.1016/j.tim.2012.04.001.
[8]Buffie CG,Pamer EG.Microbiota-mediated colonization resistance against intestinal pathogens[J].Nat Rev Immunol,2013,13(11):790-801.DOI:10.1038/nri3535.
[9]Yurist-Doutsch S,Arrieta MC,Vogt SL,et al.Gastrointestinal microbiota-mediated control of enteric pathogens[J].Annu Rev Genet,2014,48:361-382.DOI:10.1146/annurev-genet-120213-092421.
[10]Hutton ML,Mackin KE,Chakravorty A,et al.Small animal models for the study of Clostridium difficile disease pathogenesis[J].FEMS Microbiol Lett,2014,352(2):140-149.DOI:10.1111/1574-6968.12367.
[11]Reeves AE,Theriot CM,Bergin IL,et al.The interplay between microbiome dynamics and pathogen dynamics in a murine model of Clostridium difficile Infection[J].Gut Microbes,2011,2(3):145-158.DOI:10.4161/gmic.2.3.16333.
[2]Kubota H,Sakai T,Gawad A,et al.Development of Taq Manbased quantitative PCR for sensitive and selective detection of toxigenic Clostridium difficile in human stools[J].PLo S One,2014,9(10):e111684.DOI:10.1371/journal.pone.0111684.
[3]Sloan LM,Duresko BJ,Gustafson DR,et al.Comparison of realtime PCR for detection of thet cd C gene with four toxin immunoassays and culture in diagnosis of Clostridium difficile infection[J].J Clin Microbiol,2008,46(6):1996-2001.DOI:10.1128/JCM.00032-08.
[4]Best EL,Freeman J,Wilcox MH.Models for the study of Clostridium difficile infection[J].Gut Microbes,2012,3(2):145-167.DOI:10.4161/gmic.19526.
[5]Bélanger SD,Boissinot M,Clairoux N,et al.Rapid detection of Clostridium difficile in feces by real-time PCR[J].J Clin Microbiol,2003,41(2):730-734.DOI:10.1128/JCM.41.2.730-734.2003.
[6]Chen XH,Katchar K,Goldsmith JD,et al.A mouse model of Clostridium difficile-associated disease[J].Gastroenterology,2008,135(6):1984-1992.DOI:10.1053/j.gastro.2008.09.002.
[7]Britton RA,Young VB.Interaction between the intestinal microbiota and host in Clostridium difficile colonization resistance[J].Trends Microbiol,2012,20(7):313-319.DOI:10.1016/j.tim.2012.04.001.
[8]Buffie CG,Pamer EG.Microbiota-mediated colonization resistance against intestinal pathogens[J].Nat Rev Immunol,2013,13(11):790-801.DOI:10.1038/nri3535.
[9]Yurist-Doutsch S,Arrieta MC,Vogt SL,et al.Gastrointestinal microbiota-mediated control of enteric pathogens[J].Annu Rev Genet,2014,48:361-382.DOI:10.1146/annurev-genet-120213-092421.
[10]Hutton ML,Mackin KE,Chakravorty A,et al.Small animal models for the study of Clostridium difficile disease pathogenesis[J].FEMS Microbiol Lett,2014,352(2):140-149.DOI:10.1111/1574-6968.12367.
[11]Reeves AE,Theriot CM,Bergin IL,et al.The interplay between microbiome dynamics and pathogen dynamics in a murine model of Clostridium difficile Infection[J].Gut Microbes,2011,2(3):145-158.DOI:10.4161/gmic.2.3.16333.