Detection of medically important Ehrlichia by quantitative multicolor TaqMan real-time polymerase chain reaction of the dsb gene.
Ehrlichia species are the etiological agents of emerging and life-threatening tick-borne human zoonoses, in addition to causing serious and fatal infections in companion animals and livestock. We developed the first tricolor TaqMan real-time polymerase chain reaction assay capable of simultaneously detecting and discriminating medically important ehrlichiae in a single reaction. Analytical sensitivity of 50 copies per reaction was attained with templates from Ehrlichia chaffeensis, Ehrlichia ewingii, and Ehrlichia canis by amplifying the genus-specific disulfide bond formation protein gene (dsb). Ehrlichia genus-specific dsb primers amplified DNA from all known Ehrlichia species but not from other rickettsial organisms including Anaplasma platys, Anaplasma phagocytophilum, Rickettsia conorii, or Rickettsia typhi. High species specificity was attained as each species-specific TaqMan probe (E. chaffeensis, E. ewingii, and E. canis) identified homologous templates but did not cross-hybridize with heterologous Ehrlichia templates at concentrations as high as 10(8) copies. Identification of E. chaffeensis, E. ewingii, and E. canis from natural and experimental infections, previously confirmed by polymerase chain reaction and serological or microscopic evidence, demonstrated the comparable specificity and sensitivity of the dsb real-time assay. This assay provides a powerful tool for prospective medical diagnosis for human and canine ehrlichioses and for ecologic and epidemiological studies involving arthropod and mammalian hosts.
Doyle
C Kuyler
CK
Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609, USA.
Labruna
Marcelo B
MB
Breitschwerdt
Edward B
EB
Tang
Yi-Wei
YW
Corstvet
Richard E
RE
Hegarty
Barbara C
BC
Bloch
Karen C
KC
Li
Ping
P
Walker
David H
DH
McBride
Jere W
JW
D000818:Animals; D003116:Color; D004285:Dogs; D004536:Ehrlichia / Q000145:classification / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification*; D016873:Ehrlichiosis / Q000097:blood / Q000382:microbiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011490:Protein Disulfide Reductase (Glutathione) / Q000235:genetics* / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism*; D013997:Time Factors
D012336:RNA, Ribosomal, 16S; D011490:Protein Disulfide Reductase (Glutathione); D019914:Taq Polymerase