Clinical Evaluation of a New PCR Assay for Detection of Coxiella burnetii in Human Serum Samples

Abstract

A nested PCR method was developed for the detection ofCoxiella burnetii in human serum samples. Two pairs of oligonucleotide primers were designed to amplify a 438-bp fragment of the com1 gene encoding a 27-kDa outer membrane protein ofC. burnetii. The primers amplified the predicted fragments of 21 various strains of C. burnetii but did not react with DNA samples from other microorganisms. The 438-bp amplification products could be digested with restriction enzymes SspI and SalI. The utility of the nested PCR was evaluated by testing human serum samples. The com1 gene fragment was amplified from 135 (87%) of 155 indirect immunofluorescence test (IF)-positive serum samples and from 11 (11%) of 100 IF-negative serum samples. The nested PCR with primers targeted to the com1gene appeared to be a sensitive, specific, and useful method for the detection of C. burnetii in serum samples.

Differentiation of Coxiella burnetii isolates by sequence determination and PCR-restriction fragment length polymorphism analysis of isocitrate dehydrogenase gene

“…Ten serum samples from patients diagnosed as having acute Q fever [11] were used in the study. Negative controls were sera con¢rmed as having no C. burnetii by PCR with primer pairs speci¢c to the com1 gene [12].…”

“…Chromosomal DNA was extracted from puri¢ed C. burnetii and the control bacteria as described elsewhere [3]. The serum samples used for PCR were prepared as described previously [12] with some modi¢cations. Brie£y, sera (1 ml) were transferred into a 1.5-ml microcentrifuge tube and centrifuged at 13 000Ug for 40 min.…”

Detection of Coxiella burnetii specific DNA in blood samples from Japanese patients with chronic nonspecific symptoms by nested polymerase chain reaction

“…Amplimers used for C. burnetii ampli-¢cation of DNA extracted from the blood samples, generously provided by Dr. K. Hirai (Gifu University, Gifu, Japan), were 5P-AGT AGA AGC ATC CCA AGC ATT G-3P (residues 441^462; sense strand; OMP1) and 5P-TGC CTG CTA GCT GTA ACG ATT G-3P (residues 918^940; antisense strand; OMP2), designed from the nucleotide sequence of the C. burnetii com 1 gene encoding a 27-kDa OMP and 5P-GAA GCG CAA CAA GAA GAA CAC-3P (residues 475^495; sense strand; OMP3) and 5P-TTG GAA GTT ATC ACG CAG TTG-3P (residues 892^912; antisense strand; OMP4) for nested PCR. PCR using these amplimers yields a 501-and a 438-base-pair (bp) fragment [14]. The ¢rst ampli¢cation for nested PCR was performed in a total volume of 50 Wl containing 5 Wl of DNA sample, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl P , 200 WM each of dATP, dCTP, dGTP, dTTP, O.5 WM each of primers OMP1 and OMP2, and 1.25 U of Taq DNA polymerase (Takara Shuzo, Co., Ltd., Shiga, Japan).…”

“…There was no ampli¢cation of any products using the DNAs from the 14 microorganisms and negative controls. The primers OMP1-OMP2 and OMP3-OMP4 ampli¢ed the predicted products from about 5 fg of total DNA (corresponding to one organism) [14]. The samples were electrophoresed and stained with ethidium bromide (0.5 Wg/ml), visualized under UV illumination (TM-20; UVP, Inc.) at 302 nm, and photographed.…”

“…The nested PCR appeared to be a highly sensitive method for the diagnosis of C. burnetii infection [13]. Zhang et al suggested that the nested PCR is likely to be more sensitive than the IF test for antibodies to C. burnetii in the primary diagnosis of acute Q fever cases [14]. Furthermore, a Coxiella-like organism was isolated from his sera which were inoculated into A/J mice.…”