| 李伟,海荣,俞东征,张志凯,蔡虹.应用Taq Man荧光定量聚合酶链反应技术检测鼠疫耶尔森菌[J].Chinese journal of Epidemiology,2005,26(8):613-616 |
| 应用Taq Man荧光定量聚合酶链反应技术检测鼠疫耶尔森菌 |
| Establishment and application of real-time fluorescence polymerase chain reaction based on the TaqMan probes for detection of Yersinia pestis |
| Received:August 06, 2004 |
| DOI： |
| KeyWord: 鼠疫耶尔森菌 实时荧光聚合酶链反应 定量分析 |
| English Key Word: Yersinia pestis Real-time fluorescence polymerase chain reaction Quantitative analysis |
| FundProject: |
| Author Name | Affiliation | | LI Wei | Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | HAI Rong | Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | YU Dong-zheng | Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | ZHANG Zhi-kai | Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | CAI Hong | Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China |
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| Abstract: |
| 目的发展一种快速、敏感、特异的检测鼠疫耶尔森菌方法。方法应用荧光定量聚合酶链反应(PCR)技术,针对pMT1质粒上的caf1基因,pPCP1质粒上的pla基因,染色体上的与色素沉着相关的hms基因和侵袭素inv基因设计TaqMan引物探针,进行荧光定量PCR检验,评价方法的特异性、敏感性;构造并应用上述引物探针的外标对照定量;构造pla的内标对照,采用多重荧光定量PCR(FAM,VIC荧光检测),发现假阴性;采用UDG抗污染、ROX参照荧光染料矫正背景噪音,提高检验能力;检验模拟鼠疫耶尔森菌强毒株和EV菌感染的动物脾脏器样本,评价该法在实际工作的应用。结果设计的引物、探针特异性良好,pla、f1、hms指标的敏感性分别是最低检出10拷贝、1拷贝、1拷贝。结论该方法能快速、灵敏、特异检出鼠疫耶尔森菌,对鼠疫监测、预防生物恐怖等方面具有重要意义。 |
| English Abstract: |
| Objective Establishing and developing methods for quick,special and sensitive detection of Yersinia pestis(Y.pestis). Methods Using real-time fluorescence polymerase chain reaction(PCR) based on TaqMan technology with UDG anti-contamination systems and ROX reference dye, we established a method to detect Y.pestis. Probes and primers from pla,caf1, hms and inv gene were designed. An internal positive control was added to the reaction mixture in order to detect the presence of PCR inhibitors that were often found in biological samples. Sensitivity was evaluated by serial dilution of colons, internal template and Y.pestis strains while specificity was confirmed by amplifying real DNAs from bacteria, the representative Y.pestis strains and blind assay. Results The methods used could provide quick,special and sensitive detection of Y.pestis, with sensitivities of pla,f1,hms as 10, 1 and 1 copie(s) respectively. Conclusion The newly developed technologies seemed to be well suited to identify the Y.pestis in case of emergency, bio-terrorist attack and surveillance of the epidemics. |
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