A new ultrasonic high-throughput instrument for rapid DNA release from microorganisms.

Rapid detection methods for microbiological contamination are requested by many industries that respond to public health concerns. The control laboratories are replacing traditional culturing methods with faster assays based on nucleic acid amplification technologies, such as real-time PCR. However, an optimal nucleic acid sample preparation method is critical for the sensitivity and specificity of such tools. A high-throughput automated external ultrasonic device was developed for rapid lysing of microorganisms. Based on Ct values obtained from real-time PCR, there was efficient DNA release from the 16 microorganisms tested, including Gram positive and negative bacteria, bacterial spores, yeasts and spores of molds. Linearity of the lysis method was also demonstrated for Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica, Candida albicans and the spores of Aspergillus brasiliensis, with correlation coefficients (r(2)) between 0.90 and 0.98. After sonication, PCR analysis of the lysates revealed earlier Ct values (0.83 to 7.49) for S. aureus, P. aeruginosa and S. enterica compared to the bead-beating method of lysis. These results demonstrated more efficient DNA release from bacteria with the ultrasonication system. Nevertheless, for fungi, the Ct values were 0.94 to 1.61 later for sonication than for bead beating. This study demonstrates that 4min of sonication with this new automated high-throughput instrument allows for the efficient lysis of a large range of microorganisms.

Novel sample preparation method for molecular detection of Mollicutes in cell culture samples.

Research laboratories, raw materials and media suppliers as well as the biopharmaceutical industry face recurrent contamination with Mollicutes. Culture-based detection methods are very slow (28 days) and could ideally be replaced by nucleic acid testing (NAT) for rapid result. These methods are nonetheless hampered by their companion sample preparation methods. They are limited by the volume tested (0.1 to 5 mL), the protein/nucleic acid content they can accommodate and are generally performed in an open environment. The processing of low volumes of complex matrices is associated to several issues such as poor representativeness, low sensitivity, inhibition and false positives. The novel sample preparation method described in this study has been developed to overcome these limitations and to process 20-mL samples containing high loads of eukaryotic cells. A dual-membrane device is coupled to magnetic bead purification. In one single and closed device, eukaryotic cells and microorganisms are separated, contaminants are concentrated, lysed and corresponding nucleic acids are collected. This novel sample preparation method has been tested with 9 different Mollicutes. The ability to detect the contaminants down to 0.6 CFU/mL by real-time PCR among hundreds of millions of CHO-S cells (Chinese hamster ovary cells, adapted to serum-free suspension culture), without biological pre-enrichment, has been demonstrated. The novel device has been compared to manual silica spin columns, which remain the gold standard in most laboratories. These columns failed to yield the same limit of detection and reproducible results without separating mammalian cells from contaminants. Co-culture experiments have shown that the novel method allows detection of Mollicutes grown for days in presence of mammalian cells, despite the fact that these microorganisms can adhere to eukaryotic cells or invade them. The co-culture data also suggest that the novel sample preparation device might improve the live/dead cells discrimination by removing free nucleic acids from the matrix.