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柳文进, 曹晓梅, 张晓龙, 姚李四, 王海玲, 王德培. 污蝇杆菌荧光定量PCR快速检测方法建立[J]. 中国公共卫生, 2016, 32(3): 410-412. DOI: 10.11847/zgggws2016-32-03-43
引用本文: 柳文进, 曹晓梅, 张晓龙, 姚李四, 王海玲, 王德培. 污蝇杆菌荧光定量PCR快速检测方法建立[J]. 中国公共卫生, 2016, 32(3): 410-412. DOI: 10.11847/zgggws2016-32-03-43
LIU Wen-jin, CAO Xiao-mei, ZHANG Xiao-long.et al, . Establishment of Taqman real-time PCR for rapid detection of Wohlfahrtiimonas chitiniclastica bacilli[J]. Chinese Journal of Public Health, 2016, 32(3): 410-412. DOI: 10.11847/zgggws2016-32-03-43
Citation: LIU Wen-jin, CAO Xiao-mei, ZHANG Xiao-long.et al, . Establishment of Taqman real-time PCR for rapid detection of Wohlfahrtiimonas chitiniclastica bacilli[J]. Chinese Journal of Public Health, 2016, 32(3): 410-412. DOI: 10.11847/zgggws2016-32-03-43

污蝇杆菌荧光定量PCR快速检测方法建立

Establishment of Taqman real-time PCR for rapid detection of Wohlfahrtiimonas chitiniclastica bacilli

  • 摘要: 目的 污蝇杆菌(Wohlfahrtiimonas chitiniclastica bacilli)是近年新发现的条件致病菌,临床上曾引起爆发性脓毒血症,本文拟建立污蝇杆菌荧光定量PCR的快速检测方法。方法 根据污蝇杆菌W.chitiniclastica SH04的全基因组DNA序列,采用Primer Express 3.0设计引物和Taqman荧光探针,建立实时荧光定量PCR的快速检测方法,并对该方法的特异性、灵敏度和重复性进行验证。结果 污蝇杆菌荧光定量PCR快速检测方法具有良好的特异性,重复性试验的变异系数均<2%,灵敏度为19 CFU/反应(20μL反应体系),使用该方法检测6份人为污染的样品,结果均为阳性,6份阴性对照及空白对照均为阴性。结论 本研究建立的荧光定量PCR方法可以快速、准确地检测污蝇杆菌。

     

    Abstract: Objective To develop a Taqman real-time polymerase chain reaction(PCR) method for the determination of Wohlfahrtiimonas chitiniclastica(W.chitiniclastica).Methods Primers and specific fluorogenic probes(TaqMan) were designed for W.chitiniclastica with the Primer Express software based on the genome sequence of W.chitiniclastica SH04 strain; then the specificity, sensitivity, and repeatability of the method were verified.Results The method has goog specificity, with the coefficient of variation(CV) values of less than 2%in repetitive testing and a sensitivity value of 19 colony forming unit(CFU) in a 20μL reaction system.Using the method established, the detection results for 6 artificially W.chitiniclastica-polluted samples were all positive.Conclusion The real-time PCR method is a specific, sensitive and reproducible assay for the detection of W.chitiniclastica.

     

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