RESUMEN
Here we report a new real-time PCR assay using SYBR Green which provides higher sensitivity for the specific detection of low levels of Pneumocystis jirovecii. To do so, two primer sets were designed, targeting the family of genes that code for the most abundant surface protein of Pneumocystis spp., namely the major surface glycoproteins (Msg), and the mitochondrial large subunit rRNA (mtLSUrRNA) multicopy gene, simultaneously detecting two regions. PCR methods are instrumental in detecting these low levels; however, current nested-PCR methods are time-consuming and complex. To validate our new real-time Msg-A/mtLSUrRNA PCR protocol, we compared it with nested-PCR based on the detection of Pneumocystis mitochondrial large subunit rRNA (mtLSUrRNA), one of the main targets used to detect this pathogen. All samples identified as positive by the nested-PCR method were found positive using our new real-time PCR protocol, which also detected P. jirovecii in three nasal aspirate samples that were negative for both rounds of nested-PCR. Furthermore, we read both rounds of the nested-PCR results for comparison and found that some samples with no PCR amplification, or with a feeble band in the first round, correlated with higher Ct values in our real-time Msg-A/mtLSUrRNA PCR. This finding demonstrates the ability of this new single-round protocol to detect low Pneumocystis levels. This new assay provides a valuable alternative for P. jirovecii detection, as it is both rapid and sensitive.
RESUMEN
Este trabajo pretende contribuir a la caracterización de la salchicha tradicional elaborada en Tumbes por pequeños productores. Se ha obtenido información de 12 productores sobre su procedimiento de elaboración y se ha analizado la composición (pH, aw, componentes mayoritarios, ácidos láctico y acético, hidroxiprolina y estado oxidativo) y el color de 16 lotes de salchichas. Además, se ha estudiado en 6 lotes de salchichas la evolución de su composición y el momento de aparición de signos de alteración, durante 9 días de almacenamiento en las condiciones usuales de la zona (con periodos a temperatura ambiente y otros a refrigeración). La salchicha es un embutido fresco, entre húmedo y semiseco, con un contenido graso relativamente bajo y de baja acidificación. Hubo gran variabilidad entre muestras en los contenidos de componentes mayoritarios y pH. La composición cambio poco durante el almacenamiento, pero se evidenció alteración sensorial a partir del tercer día...
The aim of this work was to contribute to the characterization of a sausage produced by small-scale producers in Tumbes (Peru). A total of 12 interviews were conducted with the correspondent producers in order to gather information about the process for producing this sausage, and sixteen sausage samples were analyzed for chemical composition (pH, aw, proximal composition, lactic and acetic acids, hydroxyproline and oxidative state) and colour. Furthermore, during a 9-day storage, the changes in the sausage composition and the moment where the sausage was rejected by a sensorial panel (spoilage in odour, colour and/or texture) were assessed, using 6 branches of sausage û the storage conditions were those usually followed in the area, which included storage periods at room temperature (20-30 ºC) and under refrigeration. The sausage from Tumbes can be defined as a wet or semi-dry, (relatively) lowfat and low-acid sausage. There was observed a great variability in the proximate composition and pH among the samples analyzed. Changes in the composition and color during its storage were scarce. However, some batches of sausage were rejected by the sensory panel after three days of storage...