摘要
目的 建立一种快速、准确检测细菌中ISCR1复合型Ⅰ型整合子的MGB探针二重实时荧光PCR方法。方法 根据ISCR1复合型Ⅰ型整合子中int1基因和ISCR1元件序列设计特异性引物和探针,在多种经常携带有2种多耐药相关基因元件的细菌中检测其特异性;使用含有2种基因元件特异性序列的重组质粒标准品评价建立方法的灵敏度。结果 建立的MGB二重探针实时荧光PCR检测方法特异性好,携带2种基因元件或单独含有ISCR1或者int1的实验菌株均出现相应特异性扩增曲线,对2种基因元件均不含的菌株进行试验扩增时,均未见出现特异性扩增曲线。对2种基因元件都存在的实验菌株的扩增中,亦不存在由于2套引物/探针相互影响而造成的干扰扩增。根据标准质粒样品构建的标准曲线可确定其对int1基因和ISCR1元件的检测灵敏度分别为5.89×10~1拷贝/μl和4.13×10~1拷贝/μl。结论本研究成功建立了MGB二重探针实时荧光PCR快速检测方法。
Objective To establish a duplex MGB real-time quantitative PCR （qPCR） assay for detection of ISCR1 related complex class 1 integron in bacteria. Methods The qPCR primers and probe designing was according to intl and ISCRI sequences in ISCR1 related complex class 1 integron. The specificity and the sensitivity of this qPCR assay were evaluated by using common pathogens and target genes containing recombinant plasmids, respectively. Results The established duplex MGB real-time qPCR assay showed good specificity. The detection limit of this qPCR assay for intl and ISCR1 were determined to be 5.89 ×10^1 copies/pJ and 4. 13 × 10^1 copies/μl, respectively. Conclusion The new duplex MGB qPCR assay established in this study could be used in ISCR1 related complex class 1 integron detection and as a simple and convenient method for multi-drug resistance detection in both laboratory and large-scale surveillance.
出处
《疾病监测》
CAS
2017年第10期878-882,共5页
Disease Surveillance
基金
国家自然科学基金青年科学基金(No.31502124)
