A rapid real-time PCR/DNA resolution melting method to identify Prototheca species.

AIM: The study describes the development of a simple and rapid tool to identify yeast-like microalgae belonging to the genus Prototheca. METHODS AND RESULTS: The method, based on two-step Real Time PCR reaction followed by DNA Resolution Melting Analysis (qPCR/RMA), has been developed using reference strains belonging to both pathogenic (P. zopfii genotype 2, P. wickerhamii and P. blaschkeae) and non-pathogenic species (P. zopfii genotype 1, P. stagnora and P. ulmea). In order to validate the method, seventy recently isolated Prototheca strains were thus tested in parallel with both the first qPCR/RMA and the conventional genotype-specific PCR assay: they were classified as P. zopfii genotype 1, P. zopfii genotype 2 and P. blaschkeae, with a perfect accordance between the two above methodologies. Furthermore, we used the second qPCR/RMA to identify the other species (P. stagnora, P. ulmea and P. wickerhamii), which cannot be discriminated by conventional PCR assay. CONCLUSIONS: The assay two-step Real Time PCR is accurate, robust, cost-effective and faster than auxonographical, biochemical or conventional molecular biology methods. SIGNIFICANCE AND IMPACT OF THE STUDY: the rapid and high throughout two-step qPCR/RMA tool can be usefully used for the identification of clinical and environmental Prototheca species into the framework of the diagnosis of animal (e.g. bovine mastitis) or human protothecosis.

Mycobacterium porcinum strains isolated from bovine bulk milk: implications for Mycobacterium avium subsp. paratuberculosis detection by PCR and culture.

In this study, the isolation of 52 mycobactin-independent fast growing mycobacteria from 631 bulk milk samples (8.2%), is reported. These strains, isolated during a bulk milk survey for Mycobacterium avium subsp. paratuberculosis (Map), strongly affected Map detection both by PCR and by culture, as they gave a positive IS900 PCR signal and resulted to totally inhibit the growth of Map when spotted on HEYM slants already inoculated with 200 microl of 10-fold dilutions containing from 5 x 10 to 5 x 10(3)Map cells/ml. 16S rRNA gene sequencing, using the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems), was performed on a subset of six strains, identifying Mycobacterium porcinum with 100% homology in all six cases. The 52 strains were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene, which confirmed the identification of M. porcinum for all the isolates. Using specific primers designed on the Map-IS900 sequence and on the M. porcinum sequence determined in this study, a 1385bp sequence from the M. porcinum genome was characterized. This IS900-like sequence showed 82% homology with Map IS900. From our findings the following results emerged: (a) any culture showing one or more M. porcinum colonies represents a potential "false negative" result and should therefore be considered as contaminated; (b) IS900-like elements could be more widespread than was previously thought; (c) IS900 PCR positive results should be interpreted cautiously, as confirmed by the evidence that the primer pair used in this study resulted not to be specific.