A real-time combined polymerase chain reaction assay for the rapid detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii. 2005

Constance A Bell, and Robin Patel
Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. constance.bell@us.army.mil

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 LightCyclertrade mark 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.

UI MeSH Term Description Entries
D004269 DNA, Bacterial Deoxyribonucleic acid that makes up the genetic material of bacteria. Bacterial DNA
D004536 Ehrlichia Small, often pleomorphic, coccoid to ellipsoidal organisms occurring intracytoplasmically in circulating LYMPHOCYTES. They are the etiologic agents of tick-borne diseases of humans; DOGS; CATTLE; SHEEP; GOATS; and HORSES. Cowdria,Ehrlichia ewingii
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001426 Bacterial Proteins Proteins found in any species of bacterium. Bacterial Gene Products,Bacterial Gene Proteins,Gene Products, Bacterial,Bacterial Gene Product,Bacterial Gene Protein,Bacterial Protein,Gene Product, Bacterial,Gene Protein, Bacterial,Gene Proteins, Bacterial,Protein, Bacterial,Proteins, Bacterial
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D016873 Ehrlichiosis A tick-borne disease characterized by FEVER; HEADACHE; myalgias; ANOREXIA; and occasionally RASH. It is caused by several bacterial species and can produce disease in DOGS; CATTLE; SHEEP; GOATS; HORSES; and humans. The primary species causing human disease are EHRLICHIA CHAFFEENSIS; ANAPLASMA PHAGOCYTOPHILUM; and Ehrlichia ewingii. E chaffeensis Infection,E ewingii Infection,E. chaffeensis Infection,E. ewingii Infection,Ehrlichia Infection,Ehrlichia chaffeensis Infection,Ehrlichia ewingii Infection,Human Ehrlichiosis,E chaffeensis Infections,E ewingii Infections,E. ewingii Infections,Ehrlichia Infections,Ehrlichia chaffeensis Infections,Ehrlichia ewingii Infections,Ehrlichioses,Human Ehrlichioses,Infection, E chaffeensis,Infection, E. chaffeensis,Infections, E chaffeensis
D044366 Transition Temperature The temperature at which a substance changes from one state or conformation of matter to another. Temperature, Transition,Boiling Point Temperature,Freezing Point Temperature,Melting Point Temperature,Boiling Point Temperatures,Freezing Point Temperatures,Melting Point Temperatures,Temperature, Boiling Point,Temperature, Freezing Point,Temperature, Melting Point,Temperatures, Boiling Point,Temperatures, Freezing Point,Temperatures, Melting Point,Temperatures, Transition,Transition Temperatures
D018469 Ehrlichia chaffeensis A species of gram-negative bacteria that is the causative agent of human EHRLICHIOSIS. This organism was first discovered at Fort Chaffee, Arkansas, when blood samples from suspected human ehrlichiosis patients were studied.
D018834 Chaperonin 60 A group I chaperonin protein that forms the barrel-like structure of the chaperonin complex. It is an oligomeric protein with a distinctive structure of fourteen subunits, arranged in two rings of seven subunits each. The protein was originally studied in BACTERIA where it is commonly referred to as GroEL protein. Heat-Shock Proteins 60,hsp60 Family,GroEL Protein,GroEL Stress Protein,Heat-Shock Protein 60,hsp60 Protein,Heat Shock Protein 60,Heat Shock Proteins 60

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