Laboratory for Zoonoses and Environmental Microbiology, National Institute for Public Health and the Environment (RIVM), Anthonie van Leeuwenhoeklaan 9, Bilthoven, MA, 3721, The Netherlands.
BMC Infect Dis. 2013 Feb 14;13:86. doi: 10.1186/1471-2334-13-86.
Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance.
Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated.
A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction.
The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification.
鼻疽伯克霍尔德菌和类鼻疽伯克霍尔德菌是两种高度致病的密切相关的细菌，难以检测。致病伯克霍尔德菌在世界许多地区流行，感染病例也时有发生，这些病例有时是来自人们意想不到的地区的旅行者带来的。为了患者的治疗和流行病学监测，迫切需要快速、敏感的方法来鉴定鼻疽伯克霍尔德菌和类鼻疽伯克霍尔德菌。
根据已发表的基因组，确定了用于敏感、特异性检测致病伯克霍尔德菌的特征序列，并设计和验证了 qPCR 检测方法。
开发了一种用于检测致病伯克霍尔德菌的单反应四重 qPCR 检测方法，其中包括用于 DNA 提取和扩增内部控制的标记物。该检测方法可区分鼻疽伯克霍尔德菌和类鼻疽伯克霍尔德菌菌株，概率分析显示检测限非常低。使用多个拷贝的特征序列可以检测到每个反应少于 1 个基因组当量。
新的检测方法可快速检测致病伯克霍尔德菌，并且具有增强的灵敏度、物种分化以及包含用于 DNA 提取和 PCR 扩增的内部控制。
