Detection of Coxiella burnetii in Complex Matrices by Using Multiplex Quantitative PCR during a Major Q Fever Outbreak in The Netherlands

Abstract

ABSTRACTQ fever, caused byCoxiella 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 ofC. burnetiiDNA in veterinary and environmental matrices on suspected Q fever-affected farms. The qPCR detects threeC. burnetiitargets (icd,com1, and IS1111) and oneBacillus thuringiensisinternal control target (cry1b).Bacillus thuringiensisspores 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 targetsicd,com1, and IS1111, respectively; and no cross-reactivity with the nontarget organisms tested. Screening forC. burnetiiDNA on 29 suspected Q fever-affected farms during the Q fever epidemic in 2008 showed that swabs from dust-accumulating surfaces contained higher levels ofC. burnetiiDNA 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 theC. burnetiitargets and the internal control. The inclusion of an internal control target and threeC. burnetiitargets in one multiplex qPCR assay showed that complex veterinary and environmental matrices can be screened reliably for the presence ofC. burnetiiDNA during an outbreak.