Figure. Polymerase chain reaction (PCR) products. A. With primers for the NadA gene. Lane 1: Patient's sample with an approximately 1.02-kb product; Lane 2: Negative control; Lane 3: Positive control. Ehrlichia chaffeensis (Arkansas strain)–infected DH82 cells; Lane 4: Molecular size markers: fX174 phage DNA cleaved with HaeIII. B. With nested primers for the 120 kDa protein gene. Lane 1: Patient's sample with an approximately 1.1-kb product; Lane 2: Positive control. E. chaffeensis (Sapulpa strain)–DH82 infected cells; Lane 3: Positive control. E. chaffeensis (Arkansas strain)–infected DH82 cells; Lane 4: Molecular size markers: fX174 phage DNA cleaved with HaeIII. Amplification conditions. Ten microliters of the patient's DNA was added to a 90-µl PCR reaction tube containing 10 µl of 10X PCR buffer (Boehringer Mannheim, Indianapolis, IN), 1 µl of PCR primers ECHNADA1 and pXCR6, final concentration 1 µM each; 2 µl deoxynucleotide triphosphates (final concentration, 200 µM); and 1 µl of Taq polymerase (Boehringer Mannheim, Indianapolis, IN). Water was added to bring the volume to 100 µl. The mixture was placed in a Progene FPROGO2Y thermocycler (Techne, Princeton, NJ). A DNA lysate prepared from E. chaffeensis-infected DH82 cells was used as a positive control. The cycling program consisted of 3 min at 94°C followed by 30 cycles, each of 30 sec at 94°C, 1 min at 55°C, and 2 min at 72°C, and an additional cycle with an extension step of 3 min at 72°C. To confirm the identity of the PCR product, we sequenced the 590 bp of the nadA gene product. After amplification, the PCR products were purified by QIAquick, a PCR purification kit (QIAgen, Santa Clarita, CA). The nucleotide sequence was then determined by the dideoxynucleotide method of cycle sequencing with Taq polymerase (ABI Prism 377 DNA sequencer, Perkin-Elmer Corp., Foster City, CA). The sequencing reaction was carried out for each strand of DNA. The sequencing data were analyzed by Genetics Computer Group, Wisconsin Package software; 99.8% identity of the 590-bp overlap with nadA nucleotide sequence of Arkansas strain (Gen Bank accession number U90900) was obtained (8).