Development and evaluation of a polymerase chain reaction assay using the 16S rRNA gene for detection of Eperythrozoon suis infection

The 16S ribosomal RNA (rRNA) gene of Eperythrozoon suis was amplified using gene-specific primers developed from GenBank sequence accession U88565. Th e gene was subsequently cloned and sequenced. Based on these sequence data, 3 sets of E. suis-specific primers were designed. These primers selectivel y amplified 1394, 690, and 839 base-pair (bp) fragments of the 16S rRNA gen e from DNA of E. suis extracted from the blood of an experimentally infecte d pig during a parasitemic episode. No polymerase chain reaction (PCR) prod ucts were amplified from purified DNA of Haemobartonella felis, Mycoplasma genitalium, or Bartonella bacilliformis using 2 of these primer sets. When the primer set amplifying the 690-bp fragment was used, faint bands were ob served with H. felis as the target DNA. No PCR products were amplified from DNA that had been extracted from the blood of a noninfected pig or using P CR reagents without target DNA. The detection limits for E. suis by competi tive quantitative PCR were estimated to range from 57 and 800 organisms/ass ay. This is the first report of the utility of PCR-facilitated diagnosis an d quantitation of E. suis based on the 16S rRNA gene. The PCR method develo ped will be useful in monitoring the progression and significance of E. sui s in the disease process in the pig.