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Evaluation of filter paper to transport inactivated bacteria to detect carbapenem resistance genes by multiplex real-time PCR using high-resolution melting
Brazilian Journal of Microbiology ( IF 2.2 ) Pub Date : 2021-07-02 , DOI: 10.1007/s42770-021-00530-2
M S Carneiro 1, 2 , M N Crispim 1 , Priscila Lamb Wink 1, 2 , A L Barth 1, 2
Affiliation  

Infections caused by resistant microorganisms are a complex global public health challenge, and the way to combat the increase of resistance is the development of more modern and faster techniques for resistance detection. This study aimed to evaluate the transport of inactivated bacteria impregnated in a filter paper disk to detect carbapenem resistance genes by multiplex real-time PCR (qPCR) using high-resolution melting (HRM). A total of 88 isolates of 10 different species of Enterobacterales harboring well-characterized carbapenem resistance genes were evaluated. A full 10-µL loop of fresh growth of bacteria were impregnated in a filter paper disk, which was left at room temperature for 2 days in order to simulate the time spent in transportation. Bacterial inactivation was performed with 70% ethanol at 15 min. Afterwards, the DNA was extracted from the paper disks for further analysis by qPCR HRM. The time of 15 min in 70% ethanol was enough to inactivate all the isolates tested. It was possible to correctly identify the presence of the carbapenem resistance gene by HRM qPCR in 87 isolates (98.87%) that were transported in the filter paper disks. Our results indicated that it is possible to use filter paper to transport inactivated bacteria and to identify carbapenem resistance genes by qPCR HRM. This alternative tends to facilitate the access to this technology by many laboratories which do not have the qPCR equipment.



中文翻译:

使用高分辨率熔解的多重实时 PCR 评估滤纸转运灭活细菌以检测碳青霉烯类耐药基因

由耐药微生物引起的感染是一项复杂的全球公共卫生挑战,对抗耐药性增加的方法是开发更现代、更快的耐药性检测技术。本研究旨在通过使用高分辨率熔解 (HRM) 的多重实时 PCR (qPCR) 评估浸渍在滤纸盘中的灭活细菌的运输,以检测碳青霉烯类抗性基因。共有 10 种不同肠杆菌的 88 株分离株评估了具有良好表征的碳青霉烯类抗性基因。将一个完整的 10 µL 新鲜细菌生长环浸入滤纸盘中,将其在室温下放置 2 天,以模拟运输所花费的时间。在 15 分钟时用 70% 乙醇进行细菌灭活。之后,从纸盘中提取 DNA,通过 qPCR HRM 进行进一步分析。在 70% 乙醇中 15 分钟的时间足以灭活所有测试的分离物。通过 HRM qPCR 在滤纸盘中运输的 87 个分离物 (98.87%) 中可以正确识别碳青霉烯类抗性基因的存在。我们的结果表明,可以使用滤纸运输灭活细菌并通过qPCR HRM鉴定碳青霉烯类抗性基因。

更新日期:2021-07-02
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