Detection of Coxiella burnetii in Complex Matrices by Using Multiplex Quantitative PCR during a Major Q Fever Outbreak in The Netherlands
Detection of Coxiella burnetii in Complex Matrices by Using Multiplex Quantitative PCR during a Major Q Fever Outbreak in The Netherlands
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DOI：
10.1128/aem.05097-11
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发表时间：
2011-09-01
影响因子：
4.4
通讯作者：
Janse, I.
中科院分区：
文献类型：
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作者：
de Bruin, A.;de Groot, A.;Janse, I.
Q热是由贝氏柯克斯体（Coxiellaburnetii）引起的一种人畜共患病，在世界范围内均有分布。2007年至2009年间，荷兰东南部的大片农村地区受到Q热的严重影响。这启动了用于检测C的稳健且内部控制的多重定量PCR（qPCR）测定的开发。疑似Q热感染农场兽医和环境基质中的贝氏体DNA。qPCR检测三种C。贝氏体靶标（icd、com 1和IS 1111）和一个苏云金芽孢杆菌内部对照靶标（cry 1b）。将苏云金芽孢杆菌孢子加入样品中以控制DNA提取和PCR扩增。对qPCR检测试剂盒的性能进行了研究，结果显示效率较高;靶标icd、com 1和IS 1111的检出限分别为13.0、10.6和10.4拷贝/反应;与检测的非靶标微生物无交叉反应性。筛选C.在2008年Q热流行期间，对29个疑似Q热感染农场的贝氏体DNA进行了检测，结果表明，来自灰尘积聚表面的拭子含有较高水平的C。伯内特氏菌的DNA比山羊或绵羊的阴道拭子要多。在一些环境样品中观察到共提取物质对PCR的抑制作用，10倍或100倍稀释的样品足以获得两种C. burnetii目标和内部控制。包括内部控制目标和三个C。在一个多重qPCR测定中，贝氏体靶标显示可以可靠地筛选复杂的兽医和环境基质中是否存在C.贝氏体的DNA
Q fever, caused by Coxiella burnetii, is a zoonosis with a worldwide distribution. A large rural area in the southeast of the Netherlands was heavily affected by Q fever between 2007 and 2009. This initiated the development of a robust and internally controlled multiplex quantitative PCR (qPCR) assay for the detection of C. burnetii DNA in veterinary and environmental matrices on suspected Q fever-affected farms. The qPCR detects three C. burnetii targets (icd, com1, and IS1111) and one Bacillus thuringiensis internal control target (cry1b). Bacillus thuringiensis spores were added to samples to control both DNA extraction and PCR amplification. The performance of the qPCR assay was investigated and showed a high efficiency; a limit of detection of 13.0, 10.6, and 10.4 copies per reaction for the targets icd, com1, and IS1111, respectively; and no cross-reactivity with the nontarget organisms tested. Screening for C. burnetii DNA on 29 suspected Q fever-affected farms during the Q fever epidemic in 2008 showed that swabs from dust-accumulating surfaces contained higher levels of C. burnetii DNA than vaginal swabs from goats or sheep. PCR inhibition by coextracted substances was observed in some environmental samples, and 10- or 100-fold dilutions of samples were sufficient to obtain interpretable signals for both the C. burnetii targets and the internal control. The inclusion of an internal control target and three C. burnetii targets in one multiplex qPCR assay showed that complex veterinary and environmental matrices can be screened reliably for the presence of C. burnetii DNA during an outbreak.