A real-time combined polymerase chain reaction assay for the rapid detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii

Constance A. Bell, Robin Patel

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

A rapid real-time polymerase chain reaction (PCR) assay capable of the simultaneous detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii was developed using the LightCycler™ instrument (Roche Applied Sciences, Indianapolis, IN). The assay targets the operon groEL of the heat shock protein. Base pair mismatches in amplified DNA in regions of detection probe hybridization allowed organism differentiation by melting curve analysis. The analytical sensitivity was at least 10 copies per reaction. DNA extracts from 59 specimens previously confirmed positive for A. phagocytophilum (n = 37), E. chaffeensis (n = 19), or E. ewingii (n = 3) were used to evaluate the assay. All of the specimens positive for 1 of the 3 organisms by conventional PCR were likewise positive by the LightCycler™ method. Sensitivity and specificity were at least 100% compared with conventional PCR. This assay provides a rapid method for the detection and differentiation of the causative agents of human ehrlichiosis in the United States.

Original languageEnglish (US)
Pages (from-to)301-306
Number of pages6
JournalDiagnostic Microbiology and Infectious Disease
Volume53
Issue number4
DOIs
StatePublished - Dec 2005

Fingerprint

Ehrlichia chaffeensis
Ehrlichia
Anaplasma phagocytophilum
Real-Time Polymerase Chain Reaction
Base Pair Mismatch
Ehrlichiosis
Polymerase Chain Reaction
DNA
Operon
Heat-Shock Proteins
Freezing
Sensitivity and Specificity

Keywords

  • Anaplasma
  • Ehrlichia
  • PCR assay

ASJC Scopus subject areas

  • Infectious Diseases
  • Immunology and Allergy
  • Virology
  • Parasitology
  • Microbiology
  • Immunology
  • Applied Microbiology and Biotechnology

Cite this

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title = "A real-time combined polymerase chain reaction assay for the rapid detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii",
abstract = "A rapid real-time polymerase chain reaction (PCR) assay capable of the simultaneous detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii was developed using the LightCycler™ instrument (Roche Applied Sciences, Indianapolis, IN). The assay targets the operon groEL of the heat shock protein. Base pair mismatches in amplified DNA in regions of detection probe hybridization allowed organism differentiation by melting curve analysis. The analytical sensitivity was at least 10 copies per reaction. DNA extracts from 59 specimens previously confirmed positive for A. phagocytophilum (n = 37), E. chaffeensis (n = 19), or E. ewingii (n = 3) were used to evaluate the assay. All of the specimens positive for 1 of the 3 organisms by conventional PCR were likewise positive by the LightCycler™ method. Sensitivity and specificity were at least 100{\%} compared with conventional PCR. This assay provides a rapid method for the detection and differentiation of the causative agents of human ehrlichiosis in the United States.",
keywords = "Anaplasma, Ehrlichia, PCR assay",
author = "Bell, {Constance A.} and Robin Patel",
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AU - Bell, Constance A.

AU - Patel, Robin

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N2 - A rapid real-time polymerase chain reaction (PCR) assay capable of the simultaneous detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii was developed using the LightCycler™ instrument (Roche Applied Sciences, Indianapolis, IN). The assay targets the operon groEL of the heat shock protein. Base pair mismatches in amplified DNA in regions of detection probe hybridization allowed organism differentiation by melting curve analysis. The analytical sensitivity was at least 10 copies per reaction. DNA extracts from 59 specimens previously confirmed positive for A. phagocytophilum (n = 37), E. chaffeensis (n = 19), or E. ewingii (n = 3) were used to evaluate the assay. All of the specimens positive for 1 of the 3 organisms by conventional PCR were likewise positive by the LightCycler™ method. Sensitivity and specificity were at least 100% compared with conventional PCR. This assay provides a rapid method for the detection and differentiation of the causative agents of human ehrlichiosis in the United States.

AB - A rapid real-time polymerase chain reaction (PCR) assay capable of the simultaneous detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii was developed using the LightCycler™ instrument (Roche Applied Sciences, Indianapolis, IN). The assay targets the operon groEL of the heat shock protein. Base pair mismatches in amplified DNA in regions of detection probe hybridization allowed organism differentiation by melting curve analysis. The analytical sensitivity was at least 10 copies per reaction. DNA extracts from 59 specimens previously confirmed positive for A. phagocytophilum (n = 37), E. chaffeensis (n = 19), or E. ewingii (n = 3) were used to evaluate the assay. All of the specimens positive for 1 of the 3 organisms by conventional PCR were likewise positive by the LightCycler™ method. Sensitivity and specificity were at least 100% compared with conventional PCR. This assay provides a rapid method for the detection and differentiation of the causative agents of human ehrlichiosis in the United States.

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