环介导等温扩增(LAMP)技术检测水源病原菌

DETECTION OF WATERBORNE PATHOGENS BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP)

微生物指标是水质安全的重要指标之一,但是由于技术原因,大部分病原微生物并未被纳入水质监测中。目前各类水质标准中只有病原指示微生物的指标,然而这些指标与病原微生物的相关性并不理想。因此,开发快速简便的病原微生物检测技术是改进水质生物检测和监测的关键。环介导等温扩增技术是一种快速检测DNA且不依赖昂贵检测设备的PCR技术。尝试利用该技术检测水中的8种常见指示菌和病原菌:总大肠杆菌、沙门氏菌、鲍氏志贺氏菌、大肠埃希氏菌O157∶H7、小肠耶尔森氏菌、嗜肺军团菌、绿脓杆菌和结核杆菌,获得了针对其中5种细菌的毒力基因的特异性引物和最优等温PCR条件。环介导等温扩增技术的灵敏度可在45 min内检测出100个基因拷贝,并能在原污水中对病原菌进行定性检测。

Microbiological index is important to water safety, while the measurement of pathogenic bacteria in water is not included in the water quality monitoring due to the technique barrier. Most water quality standards contain only indicator microorganisms. However, the correlation between these indicators and real pathogens is not good enough to guarantee the safety. Therefore, developing fast and simple pathogen detection technologies is the key to improve microbiological monitoring in water. Loop-mediated isothermal amplification is a fast PCR technology to detect DNA independent of expensive PCR machine. This study attempted to detect 8 prevalent indicators and pathogenic bacteria: total coliform, Salmonella enterica, Shigella flexneri, Escherichia coli O157∶H7, Yesinia enterocolitica, Legionella pneumophila, Pseudomonas aeruginosa, Mycobacterium tuberculosis, by LAMP. The primers and PCR conditions of five of them were successfully optimized. The sensitivity of LAMP was to detect 100 gene copies within 45 minutes, and this technology can be applied in the detection of pathogens in raw wastewater.