摘要
目的本研究旨在开发一种基于多重实时定量PCR技术的快速检测方法,用于准确识别并量化口罩中常见致病菌,以评估口罩的卫生状况并控制院内感染。方法采用TaqMan探针法实时荧光定量PCR为核心技术,设计并优化特异性引物和荧光标记探针,通过一管式多重检测来同时定量三种病原菌的特异性基因序列。实验首先从NCBI数据库中筛选特异性靶基因和序列,并设计引物和探针。随后对引物和探针进行质量检测,并通过扩增特异性序列和TA克隆方法制备标准品。最终建立起标准曲线,并对口罩样本进行了检测。结果通过优化实验条件后,成功地建立了一个高效、稳定的多重实时定量PCR检测体系,并在不同浓度梯度下评估了三种病原菌标准品的检测效率。检测数据显示,引物和探针对于目标病原菌具有高度特异性,且扩增效率和信号强度均满足实验要求。在对口罩样本进行检测中,能够准确定量出其中的铜绿假单胞菌、金黄色葡萄球菌和溶血性链球菌的基因组DNA拷贝数,并据此评估口罩上的微生物污染程度。结论基于多重实时定量PCR技术的口罩中致病菌快速检测方法具有高敏感性、特异性和快速性,能够为临床和公共卫生监控提供有力的技术支撑。这种方法可以应用于医院环境的卫生监测以及对日常使用口罩的卫生状况进行评估,有助于防止传染病的传播,提升公共卫生安全。
Objective This study aims to develop a rapid detection method based on multiplex real-time quantitative PCR technology for accurate identification and quantification of common pathogenic bacteria on face masks,to assess the hygienic condition of masks and control nosocomial infections.Methods Using TaqMan probe-based real-time fluorescence quantitative PCR as the core technology,specific primers and fluorescently labeled probes were designed and optimized for simultaneous quantification of specific gene sequences from three pathogens through one-tube multiplex detection.The experiment began by screening specific target genes and sequences from the NCBI database and designing primers and probes.Subsequently,quality testing of primers and probes was conducted,and standard samples were prepared through amplification of specific sequences and TA cloning.Finally,standard curves were established,and mask samples were tested.Results After optimizing experimental conditions,an efficient and stable multiplex real-time quantitative PCR detection system was successfully established.The detection efficiency of standard samples for three pathogens was evaluated at different concentration gradients.The detection data showed that the primers and probes had high specificity for target pathogens,and the amplification efficiency and signal intensity met experimental requirements.In the detection of mask samples,the method accurately quantified the genomic DNA copy numbers of Pseudomonas aeruginosa,Staphylococcus aureus,and Streptococcus hemolyticus,thereby assessing the degree of microbial contamination on masks.Conclusion The rapid detection method for pathogenic bacteria on masks based on multiplex real-time quantitative PCR technology demonstrates high sensitivity,specificity,and speed,providing strong technical support for clinical and public health monitoring.This method can be applied to hygiene monitoring in hospital environments and assessing the hygienic condition of daily-use masks,helping to prevent the spread of infectious diseases and enhance public health safety.
作者
蒋彦洁
张辉
应珂
凌明
JIANG Yan-Jie;ZHANG Hui;YING Ke;LING Ming(Jinhua Institute for Food and Drug Inspection,Jinhua 321000,China)
出处
《实验室检测》
2024年第11期17-20,共4页
Laboratory Testing
基金
浙江省药品监管系统科技计划项目(计划编号:2021019)
口罩致病菌多重实时定量PCR快速检测技术的研究
关键词
多重实时定量PCR
口罩
微生物污染
铜绿假单胞菌
快速检测方法
multiplex real-time quantitative PCR
face masks
microbial contamination
Pseudomonas aeruginosa
rapid detection method
