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

A. de Bruin, A. de Groot, L. de Heer, J. Bok, Pieter Wielinga, M. Hamans, B. J. van Rotterdam, I. Janse

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

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.
Original languageEnglish
JournalAPPLIED AND ENVIRONMENTAL MICROBIOLOGY
Volume77
Issue number18
Pages (from-to)6516-6523
ISSN0099-2240
DOIs
Publication statusPublished - 2011
Externally publishedYes

Keywords

  • Netherlands Europe Palearctic region
  • Artiodactyla Mammalia Vertebrata Chordata Animalia (Animals, Artiodactyls, Chordates, Mammals, Nonhuman Vertebrates, Nonhuman Mammals, Vertebrates) - Bovidae [85715] sheep common host goat common host
  • Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Endospore-forming Gram-Positives [07810] Bacillus thuringiensis species spore pathogen
  • Rickettsiales Rickettsias and Chlamydias Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Rickettsiaceae [07113] Coxiella burnetii species pathogen
  • Bacillus thuringiensis cry1b gene [Endospore-forming Gram-Positives]
  • Coxiella burnetii com1 gene [Rickettsiaceae]
  • Coxiella burnetii icd gene [Rickettsiaceae] Coxiella burnetii isocitrate dehydrogenase gene
  • Coxiella burnetii IS1111 gene [Rickettsiaceae]
  • bacterial DNA
  • 03502, Genetics - General
  • 03506, Genetics - Animal
  • 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines
  • 31000, Physiology and biochemistry of bacteria
  • 31500, Genetics of bacteria and viruses
  • 36002, Medical and clinical microbiology - Bacteriology
  • 38002, Veterinary science - General and methods
  • 38006, Veterinary science - Microbiology
  • Infection
  • Molecular Genetics
  • Veterinary Medicine
  • Q fever bacterial disease etiology
  • complex matrix
  • Biochemistry and Molecular Biophysics
  • Medical Sciences
  • multiplex quantitative PCR multiplex qPCR laboratory techniques, genetic techniques
  • PCR amplification polymerase chain reaction amplification laboratory techniques, genetic techniques

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