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P. L. Goldschmidt, S. Degorge, D. Benallaoua, E. Borsali, S. Palleau, L. Batellier, C. Allouch, S. Boutboul, L. Laroche, C. Chaumeil; New Ultra-Rapid Assessment of Bacterial Contamination in Injectable Eye Solutions and Eye Drops. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3117.
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Detection of Bacteria by culture ("gold standard") for pharmaceuticals, grafts, fluids, blood and blood derivatives,medical devices, cosmetics and food requires from 18 hours to 14 days and may produce erroneous results for fastidious species.Culture are biased for Bacteria that grow only when optimal metabolic requirements can be reproduced, and detecting contamination is dependent inter alia upon statistical considerations. The goal of this work was to validate a new tool for bacterial testing.
The DNA extracted from samples and internal controls (monitor extraction-yields and absence of PCR inhibitors) in a dedicated room is introduced into 4 tubes containing primers and probes. The program for fast real-time PCR (f-real-t PCR) cosnsits in 1 x at 95°C for 10 min and 45 x 15 s at 95°C, 8 s at 52°C and 10 s at 72°C. Primers and probes: the first set detects all Bacteria, and only one pair of primers are used for the identification and quantification of Gram positive cocci and Staph, Strepto, Enterobacteria, Pseudomonas, Haemophilus and Acinetobacter selecting for each Genera probes. Additional alignments allow quantification of Corynebacteria and Propionibacteriacae.
8 Genera (Staphylococci, Streptococci, Haemophilus, Pseudomonas, Enterobacteria, Acinetobacter,Propionibacteriacae and Corynebacteria are quantified by the f-real-t PCR (≤ 0.01CFU/µl). Cultures require at least 24 hours (> 72 for Propioni) to yield results but the new f-real-t PCR 90 minutes, with 100% specificity and no false negatives. Larger series should be tested to confirm the usefulness of this test for routine bacterial sterility controls.
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