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New test for rapid Acanthamoeba diagnosis
Author(s) -
GOLDSCHMIDT P,
DEGORGE S,
BENALLAOUA D,
BORSALI E,
LE BOUTER A,
BATELLIER L,
BORDERIE V,
LAROCHE L,
CHAUMEIL C
Publication year - 2011
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2011.2343.x
Subject(s) - primer (cosmetics) , biology , microbiology and biotechnology , polymerase chain reaction , acanthamoeba , false positive paradox , chemistry , genetics , organic chemistry , machine learning , computer science , gene
Purpose Acanthamoeba keratitis (AK) is a sight‐threatening infection. Classic PCR enhanced sensitivity but required post‐amplification procedures, increasing contamination risks. The reported real‐time PCRs are unable to detect all the genotypes associated with pathology. We present a new strategy validated American Type Cell Collection strains and corneal scrapings. Methods A were detected by a fast PCR (f‐d‐real‐t PCR) and negativity confirmed by SYBR Green. Sequences selected in the mitochondrion were: forward primer: 5‘GCAGTCGCGGTAATACGA; reverse: 5’ACCACCTACGCACCCTTTACA and probe: 6‐FAM‐AGTGTTATTCGCATTGACTGGGTGTAA‐TAMRA. Results The new test detects all A with different lab equipment (1 cyst or less/specimen). A.astronyxis signals are inferior to others; however, primers diluted in SYBR Green mix (without probe) detect 0.1 cyst/µl or less of this strain. For clinical samples microscopic examination and cultures detected 6 out 10 but f‐d‐real‐t PCR 100% with results confirmed by SYBR Green. Other PCRs bracketing different regions (ribosome) detected 80 % and produced false positives for samples containing Salmonella lexinton, C. sporulans, S. marcescens and Propionibacteria. Conclusion Highly sensitive diagnosis is necessary to administrate efficient treatments at the onset of AK. The strains from the ATCC with higher sensitivity and specificity than techniques previously reported. New approaches based on High Resolution Melting r‐t PCR are in development in the CHNO to detect and molecularly characterize in one run different strains of protozoa and Fungi infecting the eye.