Rapid detection and simultaneous molecular profile characterization of Acanthamoeba infections

Abstract Diagnosis of Acanthamoeba by microscopic examination, culture, and polymerase chain reactions (PCRs) has several limitations (sensitivity, specificity, lack of detection of several strains, cost of testing for discrimination among strains). We developed a new high-resolution melting real-ti...

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Bibliographic Details
Published in:Diagnostic microbiology and infectious disease 2012-10, Vol.74 (2), p.137-141
Main Authors: Goldschmidt, Pablo, Degorge, Sandrine, Benallaoua, Djida, Batellier, Laurence, Di Cave, David, Chaumeil, Christine
Format: Article
Language:English
Subjects:
Acanthamoeba
Acanthamoeba - isolation & purification
Amebiasis - diagnosis
Amebiasis - parasitology
Biological and medical sciences
Cornea - parasitology
Detection
Fundamental and applied biological sciences. Psychology
Genotype
High-resolution melting real-time PCR
Humans
Infectious Disease
Infectious diseases
Internal Medicine
Keratitis
Medical sciences
Microbiology
Molecular Diagnostic Techniques - economics
Molecular Diagnostic Techniques - methods
Parasitology - economics
Parasitology - methods
Real-Time Polymerase Chain Reaction - economics
Real-Time Polymerase Chain Reaction - methods
Sensitivity and Specificity
Time Factors
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Summary:Abstract Diagnosis of Acanthamoeba by microscopic examination, culture, and polymerase chain reactions (PCRs) has several limitations (sensitivity, specificity, lack of detection of several strains, cost of testing for discrimination among strains). We developed a new high-resolution melting real-time PCR (HRM) to detect and characterize Acanthamoeba infections. HRM performances were evaluated with strains from the American Type Culture Collection (ATCC) and with 20 corneal scrapings. The DNA extracted from specimens were amplified, detected, and characterized in 1 run using 2 original primers diluted in a solution containing an intercalating dye. Detection and molecular characterization of Acanthamoeba infections could be achieved in less than 2.5 h with a dramatic reduction in cost of reactants (postamplification procedures and radioactive or fluorescent-labeled molecular probes were unnecessary). HRM detection limits were 0.1 cyst/μL or less (including genotypes T5 and T11), and its sensitivity and specificity were higher than other molecular tests. For the tested strains from the ATCC, the HRM drafted 4 different profiles: Type I (genotypes T2 and T4), Type II (T5 and T7), Type III (T8), and Type IV (T1, T3, T6, T9, T11, T12, and T13).
ISSN:0732-8893
1879-0070
DOI:10.1016/j.diagmicrobio.2012.06.001