中华流行病学杂志
中華流行病學雜誌
중화류행병학잡지
CHINESE JOURNAL OF EPIDEMIOLOGY
2012年
2期
226-228
,共3页
泰泽氏菌%小沟结合物探针%荧光定量PCR
泰澤氏菌%小溝結閤物探針%熒光定量PCR
태택씨균%소구결합물탐침%형광정량PCR
Clostridium piliforme%Minor groove binder probe%Fluorescence quantitative PCR
目的 建立泰泽氏菌的TaqMan小沟结合物(MGB)探针荧光定量PCR检测方法.方法 针对泰泽氏菌16S rRNA基因的保守区设计特异性引物和探针,建立MGB探针荧光定量PCR方法,并验证该方法的特异性、敏感性和稳定性.对2008-2011年采集的1156份临床样本进行检测,同时进行普通PCR检测作为对照.结果 泰泽氏菌TaqMan MGB探针荧光定量PCR方法具有高度特异性,与肝螺杆菌、幽门螺杆菌、空肠弯曲菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌之间无交叉反应,检测灵敏度达2.2 copy/μl.标准曲线显示各浓度范围内具有良好线性关系,相关系数为0.999,斜率为-3.204,PCR效率为100％.对1156份临床样本进行检测,荧光定量PCR检出101份泰泽氏菌阳性样本,而普通PCR则仅检出44份.荧光定量PCR方法从临床样本中检出泰泽氏菌DNA仅需2h.结论 TaqMan MGB探针荧光定量PCR方法具有特异、灵敏和稳定性,适于泰泽氏菌的快速检测.
目的 建立泰澤氏菌的TaqMan小溝結閤物(MGB)探針熒光定量PCR檢測方法.方法 針對泰澤氏菌16S rRNA基因的保守區設計特異性引物和探針,建立MGB探針熒光定量PCR方法,併驗證該方法的特異性、敏感性和穩定性.對2008-2011年採集的1156份臨床樣本進行檢測,同時進行普通PCR檢測作為對照.結果 泰澤氏菌TaqMan MGB探針熒光定量PCR方法具有高度特異性,與肝螺桿菌、幽門螺桿菌、空腸彎麯菌、侵肺巴斯德氏菌、大腸埃希菌、銅綠假單胞菌之間無交扠反應,檢測靈敏度達2.2 copy/μl.標準麯線顯示各濃度範圍內具有良好線性關繫,相關繫數為0.999,斜率為-3.204,PCR效率為100％.對1156份臨床樣本進行檢測,熒光定量PCR檢齣101份泰澤氏菌暘性樣本,而普通PCR則僅檢齣44份.熒光定量PCR方法從臨床樣本中檢齣泰澤氏菌DNA僅需2h.結論 TaqMan MGB探針熒光定量PCR方法具有特異、靈敏和穩定性,適于泰澤氏菌的快速檢測.
목적 건립태택씨균적TaqMan소구결합물(MGB)탐침형광정량PCR검측방법.방법 침대태택씨균16S rRNA기인적보수구설계특이성인물화탐침,건립MGB탐침형광정량PCR방법,병험증해방법적특이성、민감성화은정성.대2008-2011년채집적1156빈림상양본진행검측,동시진행보통PCR검측작위대조.결과 태택씨균TaqMan MGB탐침형광정량PCR방법구유고도특이성,여간라간균、유문라간균、공장만곡균、침폐파사덕씨균、대장애희균、동록가단포균지간무교차반응,검측령민도체2.2 copy/μl.표준곡선현시각농도범위내구유량호선성관계,상관계수위0.999,사솔위-3.204,PCR효솔위100％.대1156빈림상양본진행검측,형광정량PCR검출101빈태택씨균양성양본,이보통PCR칙부검출44빈.형광정량PCR방법종림상양본중검출태택씨균DNA부수2h.결론 TaqMan MGB탐침형광정량PCR방법구유특이、령민화은정성,괄우태택씨균적쾌속검측.
Objective To develop a TaqMan MGB probe-based,sensitive and specific fluorescence quantitative PCR assay method for rapid detection of Clostridium piliforme.Methods Primers and probes specific to 16S rRNA gene of Clostridium piliforme were designed.A TaqMan MGB probe-based,fluorescence quantitative PCR method was established.Specificity,sensitivity and stability of the method were assessed,followed by real-time quantitative PCR assay to detect Clostridium piliforme on 1156 clinical specimens during 2008-2011 and compared with conventional PCR assay.Results The specificity of TaqMan MGB probe-based fluorescence quantitative PCR was high and did not show cross-reactivity with Helicobacter hepaticus,Helicobacter pylori,Campylobacter jejuni,Pasteurella pneumotropica,Escherichia coli or Pseudomonas aeruginosa.The detection limit was 2.2 copies/μl.The correlation coefficient and slope value of standard curve were 0.999 and -3.204,respectively and the efficiency of TaqMan MGB-based probe fluorescence quantitative PCR assay was 100％.When the TaqMan MGB-based probe fluorescence quantitative PCR assay was preformed to detect Clostridium piliforme on 1156 clinical specimens,a total of 101 specimens showed positive on Clostridium piliforme.However,only 44 specimens showed positive when conventional PCR was used.The real-time quantitative PCR for Clostridium piliforme could be completed within 2 hours.Conclusion The TaqMan MGB-based probe fluorescence quantitative PCR assay method was a reliable,specific,sensitive and useful tool for rapid detection of Clostridium piliforme.
