Development, Optimization, and Validation of a Multiplex Real-Time PCR Assay on the BD MAX Platform for Routine Diagnosis of Acanthamoeba Keratitis
| Autor: | Claire Richarme, Danièle Maubon, Diane Bernheim, Cécile Garnaud, Lucie Post, Marie Gladys Robert |
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| Přispěvatelé: | CHU Grenoble, Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF) |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
medicine.medical_specialty Genotype [SDV]Life Sciences [q-bio] Biopsy Acanthamoeba Real-Time Polymerase Chain Reaction Sensitivity and Specificity Pathology and Forensic Medicine Keratitis Cornea 03 medical and health sciences 0302 clinical medicine medicine Humans Multiplex biology business.industry Diagnostic Tests Routine Becton dickinson Reproducibility of Results DNA Protozoan medicine.disease biology.organism_classification DNA extraction Dermatology 3. Good health 030104 developmental biology Real-time polymerase chain reaction Acanthamoeba keratitis Acanthamoeba Keratitis Molecular Diagnostic Techniques 030220 oncology & carcinogenesis Corneal scrapings Molecular Medicine business Multiplex Polymerase Chain Reaction |
| Zdroj: | Journal of Molecular Diagnostics Journal of Molecular Diagnostics, American Society for Investigative Pathology (ASIP), 2020, 22, pp.1400-1407. ⟨10.1016/j.jmoldx.2020.09.001⟩ |
| ISSN: | 1943-7811 1525-1578 |
| DOI: | 10.1016/j.jmoldx.2020.09.001⟩ |
| Popis: | The reported number of cases of Acanthamoeba amebic keratitis (AK) is continually increasing. Molecular diagnosis has become the first choice of ophthalmologists for identifying and confirming this clinically problematic diagnosis. However, in-house molecular diagnostic procedures are time-consuming and may not be compatible with the urgency of the situation. In this study, a previous in-house AK-PCR technique was adapted for use on BD MAX (Becton Dickinson, Heidelberg, Germany), a fully integrated, automated platform for molecular biology, for the rapid routine diagnosis of AK. Different protocols were compared to optimize DNA extraction from Acanthamoeba cysts. The analytical parameters of the AK-BD MAX PCR were evaluated. Thirty-two samples were simultaneously tested with AK-BD MAX PCR and the original AK-PCR from which it was developed. A thermal-shock pretreatment protocol was validated. The analytical parameters obtained with BD MAX were similar to those obtained with the previous in-house AK-PCR method. The performance of AK-BD MAX PCR was then assessed for routine testing on 40 clinical samples, mostly corneal scrapings. Frozen, ready-to-use, in-house PCR premixes were stable over 8 months. Overall, 34 of the 40 clinical samples (85%) were considered to be true negatives; 4 (10%), probable AK; and 2 (5%), possible AK. This newly developed AK-BD MAX PCR is reliable, rapid, and efficient, and should facilitate Acanthamoeba keratitis diagnosis. |
| Databáze: | OpenAIRE |
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