A real-time PCR assay for detection of the Ehrlichia muris-like agent, a newly recognized pathogen of humans in the upper Midwestern United States.
The Ehrlichia muris-like agent (EMLA) is an emerging, tick-transmitted human pathogen that occurs in the upper Midwestern United States. Here, we describe the development and validation of a p13-based quantitative real-time PCR TaqMan assay to detect EMLA in blood or tissues of ticks, humans, and rodents. The primer and probe specificities of the assay were ascertained using a large panel of various Ehrlichia species and other members of Rickettsiales. In addition to control DNA, both non-infected and EMLA-infected human blood, Mus musculus blood, and M. musculus tissue extracts were evaluated, as were non-infected and EMLA-infected Ixodes scapularis and uninfected Dermacentor variabilis DNA lysates. The specificity of the probe was determined via real-time PCR. An EMLA p13 control plasmid was constructed, and serial dilutions were used to determine the analytical sensitivity, which was found to be 1 copy per 4μl of template DNA. The sensitivity and specificity of this assay provides a powerful tool for ecological studies involving arthropod vectors and their mammalian hosts.
Rickettsial Zoonoses Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Atlanta, GA 30329, United States. Electronic address: wro8@cdc.gov.
Michelle E J
MEJ
Allerdice
Division of Clinical Microbiology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, United States.
Bobbi S
BS
Pritt
Division of Clinical Microbiology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, United States.
Lynne M
LM
Sloan
Rickettsial Zoonoses Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Atlanta, GA 30329, United States.
Christopher D
CD
Paddock
Rickettsial Zoonoses Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Atlanta, GA 30329, United States.
Sandor E
SE
Karpathy
D004536:Ehrlichia / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016873:Ehrlichiosis / Q000453:epidemiology* / Q000382:microbiology*; D006801:Humans; D015146:Midwestern United States; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
Ehrlichia muris; Ehrlichia muris-like (EML) agent; Ehrlichiosis; Tick-borne disease