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志贺菌6种毒力基因的多重PCR检测 被引量:1
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作者 徐兰英 许汴利 马宏 《郑州大学学报(医学版)》 CAS 北大核心 2009年第6期1218-1221,共4页
目的:建立志贺菌6种毒力基因[志贺菌肠毒素1基因(set1)、志贺菌肠毒素2基因(sen)、侵袭性质粒抗原H基因(ipaH)、侵袭相关蛋白基因(ial)、志贺毒素1基因(stx1)及调控基因(virA)]的多重PCR鉴定方法,实现多基因的同时检测。方法:选择志贺菌... 目的:建立志贺菌6种毒力基因[志贺菌肠毒素1基因(set1)、志贺菌肠毒素2基因(sen)、侵袭性质粒抗原H基因(ipaH)、侵袭相关蛋白基因(ial)、志贺毒素1基因(stx1)及调控基因(virA)]的多重PCR鉴定方法,实现多基因的同时检测。方法:选择志贺菌的6种毒力基因作为靶基因,以A群和C群4株标准及B群和D群4株临床分离株为模型,应用降落PCR方法在1个反应管中同时进行扩增,根据扩增片段大小定性判断结果。通过样本倍比稀释检验多重PCR的灵敏性,并在NCBI网站上用BLAST检索各引物的特异性。另选取河南省2001年至2007年115株志贺菌进行多重PCR检测,以验证多重PCR的可行性。结果:多重PCR能同时检测上述6种毒力基因,与单基因PCR分别检测6种毒力基因相比具有相近的灵敏度与特异性。应用多重PCR方法检测115株志贺菌,除stx1外,其余5种毒力基因的阳性率均在85%以上。结论:多重PCR鉴定志贺菌毒力基因具有简便、快速的特点,适用于毒力基因鉴定和流行病学调查研究。 展开更多
关键词 志贺 多重PCR 志贺肠毒素1基因 志贺菌肠毒素2基因 侵袭性质粒抗原H基因 侵袭相关蛋白基因 志贺毒素1基因 调控基因
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Establishment and Application of a Real-time PCR Method for Detecting stx2 Gene in Shiga Toxin-producing Escherichia coli(STEC)
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作者 汪伟 张雪寒 +6 位作者 王润 何孔旺 温立斌 倪艳秀 周俊明 王小敏 李彬 《Agricultural Science & Technology》 CAS 2014年第9期1473-1477,共5页
[Objective] This study aimed to establish a real-time PCR method for de- tecting stx2 gene in Shiga toxin-producing E. coli (STEC). [Method] According to the known STEC stx2 gene sequences published in GenBank, PCR ... [Objective] This study aimed to establish a real-time PCR method for de- tecting stx2 gene in Shiga toxin-producing E. coli (STEC). [Method] According to the known STEC stx2 gene sequences published in GenBank, PCR primers and probes were designed based on the conserved region to construct recombinant plasmid as a positive template, thus optimizing the reaction conditions and establishing the real- time PCR method. [Result] A standard curve was established based on the opti- mized real-time PCR system, indicting a good linear correlation between the initial template concentration and Ct value, with the correlation coefficient F^e of above 0.995. The established method had a good specificity, without non-specific amplifica- tion for 10 non-STEC intestinal bacterial strains; the detection limit of initial template was 1.0x102 copies/μI, indicating a high sensitivity; furthermore, the coefficients of variation within and among batches were lower than 1% and 5% respectively, sug- gesting a good repeatability. [Conclusion] In this study, a real-time PCR method was successfully established for detecting STEC stx2 gene, which provided technical means for rapid detection of STEC in samples. 展开更多
关键词 Shiga toxin-producing E. colr Shiga toxin 2 gene Real-time PCR
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