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).

Goldschmidt, P., Degorge, S., Benallaoua, D., Batellier, L., DI CAVE, D., Chaumeil, C. (2012). Rapid detection and simultaneous molecular profile characterization of Acanthamoeba infections. DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE, 74(2), 137-141 [10.1016/j.diagmicrobio.2012.06.001].

Rapid detection and simultaneous molecular profile characterization of Acanthamoeba infections

DI CAVE, DAVID;
2012

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).
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore VET/06 - Parassitologia e Malattie Parassitarie degli Animali
English
Acanthamoeba; Amebiasis; Cornea; Humans; Molecular Diagnostic Techniques; Parasitology; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Time Factors
Goldschmidt, P., Degorge, S., Benallaoua, D., Batellier, L., DI CAVE, D., Chaumeil, C. (2012). Rapid detection and simultaneous molecular profile characterization of Acanthamoeba infections. DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE, 74(2), 137-141 [10.1016/j.diagmicrobio.2012.06.001].
Goldschmidt, P; Degorge, S; Benallaoua, D; Batellier, L; DI CAVE, D; Chaumeil, C
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/127382
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