Abstract. A nested polymerase chain reaction specific for Ehrlichia chaffeensis was used to attempt to amplify DNA from extracts of 100 individual ticks collected from 13 counties in central Missouri. Seventeen of 59 Amblyomma americanum and six of 41 Dermacentor variabilis ticks exhibited the characteristic 389-basepair product. This supports the hypothesis that these tick species may be vectors of human monocytic ehrlichiosis.Human monocytic ehrlichiosis is a nonspecific febrile illness first described in 1987. 1 Based on epidemiologic and polymerase chain reaction (PCR) studies, the etiologic agent, Ehrlichia chaffeensis, is believed to be transmitted predominantly by the Lone Star tick, Amblyomma americanum. 2,3 Missouri is one of the leading states for reported cases of human monocytic ehrlichiosis.The PCR has been used to demonstrate the presence of the 16S rRNA gene sequence of E. chaffeensis in extracts of pooled ticks and in individual ticks. 3,4 The former method used DNA extracted from a pool of ticks to potentially increase target DNA sequences, while the latter was felt to lack sensitivity.A nested PCR greatly enhances sensitivity of detection of target nucleic acid sequences. 5 This method has been used to increase the sensitivity for detection of E. chaffeensis in the blood of white-tailed deer. 6 We now report the results of applying an E. chaffeensis-specific nested PCR technique to 100 individual ticks of the species A. americanum and Dermacentor variabilis collected from 13 counties in Missouri. MATERIALS AND METHODSTicks were collected during the spring and summer of 1995 from 13 different counties in Missouri (Figure 1). Sources of the ticks included vegetation, domestic animals, and humans. The species, sex, and stage of development of each tick were identified by an entomologist; the sex of nymphal ticks could not be determined by visual inspection.DNA was extracted from ticks by a modification of a method previously described. 4 Briefly, individual ticks were placed in 200 l of water, crushed, and boiled for 10 min. This material was diluted 1:2 in 10 mM Tris, pH 8.0, 10 mM NaCl and lysed in the presence of 1.0% sodium dodecyl sulfate and 100 ng of proteinase K/ml for 2 hr at 50ЊC. The lysed suspension was extracted twice with an equal volume of phenol-chloroform. A 1:10 dilution of 3 M sodium acetate, pH 5.5, and a 1:1,000 dilution of 20 mg/ml of glycogen (Boehringer Mannheim, Indianapolis, IN) were added to the extracted aqueous phase. The DNA was precipitated with cold ethanol, washed once with 70% ethanol, dried, and resuspended in 40 l of water.The DNA templates from individual ticks were examined by PCR by a modification of a method previously described. 6 For the initial amplification, 10 l of each template sample was amplified in a 60-l reaction mixture containing the primers ECB (5Ј-CGTATTACCGCGGCTGCTGGCA-3Ј) and EG1 (5Ј-CTCAGAACGAACGCTGGCGG-3Ј) and GeneAmp reagents (Perkin-Elmer Cetus, Norwalk, CT) at concentrations described in the GeneAmp protocol. ECB is part of the primer se...

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Open sandwich (OS) immunoassay utilizes antigen-dependent stabilization of an antibody variable region to quantify various antigens, enabling noncompetitive detection of small molecules with a broad working range. For further improvement of its sensitivity, OS Immuno-PCR was attempted with recombinant fusion proteins. The maltose binding protein-fused heavy chain variable region (MBP-V(H)) of an antibody that recognizes the C-terminal fragment of human osteocalcin (bone Gla protein, BGP), a biomarker for bone-related diseases, was immobilized onto microplate wells, and the antigen together with streptavidin (SA)-fused light chain variable region of the same antibody (SA-V(L)) was added and incubated. The amount of immobilized SA-V(L) was quantified by tethered biotinylated DNA, which was used to estimate the amount of antigen by realtime PCR. When BGP C-terminal peptide was detected, the limit of detection was 100 fg/mL, which was superior than that of our previously reported phage-based OS Immuno-PCR. The developed OS Immuno-PCR system will be useful for the detection of small molecule biomarkers for disease prevention.