National Institute for Public Health and the Environment, Laboratory for Zoonoses and Environmental Microbiology, Bilthoven, The Netherlands.
BMC Microbiol. 2010 Dec 8;10:314. doi: 10.1186/1471-2180-10-314.
Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples.
Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains.
The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum.
如果不能及时发现，一些病原体可能会严重影响公众健康。为了减少这些高致病性微生物的影响，需要快速、准确的诊断工具来检测各种样本中的病原体，包括环境样本。
设计了多重实时 PCR 来快速可靠地检测三种可能导致人类高发病率和死亡率的主要病原体：炭疽杆菌、土拉弗朗西斯菌和鼠疫耶尔森菌。所开发的检测方法可检测三个病原体特异性靶标，包括至少一个染色体靶标，以及一个来自苏云金芽孢杆菌的靶标，该靶标用作核酸提取的内参，以及成功的 DNA 扩增。PCR 的验证表明具有高分析灵敏度、特异性和对各种病原体菌株的覆盖范围。
所开发的多重 qPCR 检测方法允许同时快速检测 3 种病原体特异性靶标，而不会降低灵敏度。将苏云金芽孢杆菌孢子用作内参进一步减少了假阴性结果。这确保了高度可靠的检测，同时将模板消耗和实验室工作量保持在最低水平。
