Enhanced Detection of Coxiella burnetii with a Complementary Locked Primer-Based Real-Time PCR Method
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Mol Diagn Ther 2011; 15 (2): 103-107 1177-1062/11/0002-0103/$49.95/0
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Enhanced Detection of Coxiella burnetii with a Complementary Locked Primer-Based Real-Time PCR Method Eun-Ju Kim,1 Hong Yong Kang,1 Kyu-Jam Hwang,1 Sang-Hee Park,1 Mi-Yeoun Park,1 Sungdo Park,1 Jin Seok Yu,2 Ji Sung Park,2 Sang Hyeon Kang2 and Hyuk Chu1 1 Division of Zoonoses, Center for Immunology and Pathology, National Institute of Health, Korea Centers for Disease Control & Prevention, Chungcheongbuk-do, Korea 2 iNtRON Biotechnology, Seongnam, Gyeonggi-do, Korea
Abstract
Background and Objective: Coxiella burnetii is the bacterial causative agent of Q fever in humans. Because Q fever can establish itself with an initial inoculation of fewer than ten C. burnetii cells, a sensitive detection method for C. burnetii infection is needed for early detection. We aimed to evaluate the effectiveness of a complementary locked primer (CLP)-based real-time PCR method for sensitive detection of C. burnetii infection. Methods: To evaluate the ability of CLPs to enhance the efficiency of the real-time PCR assay for the C. burnetii IS1111 insertion sequence, the mean threshold cycle values from 20 real-time PCR replicates with either CLPs or conventional primers were determined using tenfold serial dilutions (102–108) of purified C. burnetii Nine Mile genomic DNA. In addition, the cross-reactivity between C. burnetii and 31 nonCoxiella species was examined. Results: The CLP-based real-time PCR allowed specific and reliable detection of as few as 59 copies of the IS1111 element present in the genome of C. burnetii, which represents approximately 2.96 genome equivalents or three cells of C. burnetii. These results demonstrate the effectiveness of CLP-based real-time PCR for sensitive detection of C. burnetii infection. Conclusion: It can be concluded that the CLP-based real-time PCR assay is a more appropriate method for sensitive detection and quantification of C. burnetii than previously reported methods.
Coxiella burnetii is the causative agent of Q fever, and it causes infections worldwide in both humans and domestic mammals, such as sheep, goats, and cattle.[1-4] In humans, Q fever can manifest as an acute infection characterized by a flu-like febrile illness, headache, and atypical pneumonia.[1,5] The serious chronic form of the disease results in endocarditis.[1] C. burnetii infection mainly occurs through inhalation of aerosols, and the disease can establish itself with an initial inoculation of fewer than ten cells.[6,7] Moreover, it has been reported that only one C. burnetii cell is required to produce infection under experimental conditions.[5] Because of this low inoculum, C. burnetii is considered a potential bioterrorist agent and is classified as a Category B biological terrorist agent by the Centers for Disease Control and Prevention (CDC).[5,8]
The standard diagnosis of Q fever relies on an indirect immunofluorescence assay.[1] Because antibodies are absent in the early phases of the
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