Development of a genomic DNA reference material for Salmonella enteritidis detection using polymerase chain reaction.

Several rapid methods based on nucleic acids can detect foodborne pathogens, such as Salmonella spp. However, a common reference that enables metrological traceability among measurement results is not available. Reference materials (RM) are thus key to guarantee methodological comparability. This study developed a candidate genomic DNA reference material for Salmonella enteritidis quantification to establish performance conditions and reference values for normalized RM production. The growth of Salmonella enteritidis ATCC® 13076 in Rappaport Vassiliadis selective medium was characterized, and we optimized a method of DNA extraction using cetrimonium bromide (CTAB) and LiCl. In a first stage six concentrations of DNA were prepared with and without yeast RNA (40 ng/µL) to evaluate its effect as a stabilizer in terms of homogeneity and short-term stability. Based on the findings, in a second stage two DNA concentrations were prepared and a reference value with its uncertainty was assigned based on the results of characterization, homogeneity, and stability studies using digital polymerase chain reaction and the gene targets, invA, ttr, and hilA. The material was stable for 9 months at 4 °C, with a expanded uncertainty contribution range of 11%-14%. The novel candidate RM is the first to be developed nationwide and will improve the quality of measurements in the area of food safety.

Validation of Droplet Digital Polymerase Chain Reaction for Salmonella spp. Quantification.

Salmonellosis is a foodborne disease caused by Salmonella spp. Although cell culture is the gold standard for its identification, validated molecular methods are becoming an alternative, because of their rapidity, selectivity, and specificity. A simplex and duplex droplet digital polymerase chain reaction (ddPCR)-based method for the identification and quantification of Salmonella using ttr, invA, hilA, spaQ, and siiA gene sequences was validated. The method has high specificity, working interval between 8 and 8,000 cp/µL in ddPCR reaction, a limit of detection of 0.5 copies/µL, and precision ranging between 5 and 10% measured as a repeatability standard deviation. The relative standard measurement uncertainty was between 2 and 12%. This tool will improve food safety in national consumption products and will increase the competitiveness in agricultural product trade.

The P-type ATPase CtpG preferentially transports Cd2+ across the Mycobacterium tuberculosis plasma membrane.

P1B-type ATPases are involved in heavy metal transport across the plasma membrane. Some Mycobacterium tuberculosis P-type ATPases are induced during infection, suggesting that this type of transporter could play a critical role in mycobacterial survival. To date, the ion specificity of M. tuberculosis heavy metal-transporting P1B-ATPases is not well understood. In this work, we observed that, although divalent heavy metal cations such as Cu2+, Co2+, Ni2+, Zn2+ Cd2+ and Pb2+ stimulate the ATPase activity of the putative P1B-type ATPase CtpG in the plasma membrane, whole cells of M. smegmatis expressing CtpG only tolerate high levels of Cd2+ and Cu2+. As indicator of the catalytic constant, Michaelis-Menten kinetics showed that CtpG embedded in the mycobacterial cell membrane has a V max/K m ratio 7.4-fold higher for Cd2+ than for Cu2+ ions. Thus, although CtpG can accept different substrates in vitro, this P-type ATPase transports Cd2+ more efficiently than other heavy metal cations across the mycobacterial plasma membrane.

Evaluación de la actividad citotóxica y leishmanicida de extractos y fracciones de Piper cumanense y Piper holtonii / Evaluation of the cytotoxic and leishmanicidal activity of extracts and fractions of Piper cumanense and Piper holtonii

Especies del género Piper son reportadas como promisorias para el tratamiento de enfermedades tropicales. Este estudio evalúa la actividad citotóxica y leishmanicida de extractos y fracciones de diferente polaridad obtenidas de las especies vegetales Piper cumanense (P. cumanense) y Piper holtonii (P. holtonii); se emplearon macrófagos murinos J774 y promastigotes de Leishmania panamensis MHOM/CO/87/UA140. La fracción hexánica (PcH) presentó un efecto leishmanicida con una selectividad de 2 en los modelos in vitro empleados. Esta selectividad permite sugerir una potencial actividad antileishmanial, que amerita seguir siendo explorada.

Piper genus’ species are reported as promissory as tropical diseases treatment. This research showed the cytotoxic and leishmanicidal activity of extracts and fractions of different polarity derived from Piper cumanense (P. cumanense) and Piper holtonii (P. holtoni) on murine macrophages J774 and L. panamensis promastigotes (MHOM/ CO/87/UA140). Hexanic fraction (PcH) exhibited leishmanicidal effect with 2-fold index selectivity in this in vitro model used. These results suggest a potential antileishmanial activity which should be more studied.