Detección directa de genes toxigénicos de Bacillus cereus en fécula de maíz y harina de trigo mediante reacción en cadena de la polimerasa múltiple (mPCR) / Direct detection of toxigenic genes of Bacillus cereus in corn starch and wheat flour using multiplex polymerase chain reaction (mPCR)

Introducción: Bacillus cereus es una bacteria contaminante de alimentos y patógena en humanos, cuya toxina emética o cereúlida (Ces) causa el síndrome emético y las enterotoxinas hemolítica o hemolisina BL (Hbl), no hemolítica (Nhe) y citotoxina K (CytK), el síndrome diarreico. Objetivo: Determinar la presencia de genes toxigénicos de Bacillus cereus en muestras de ADN obtenido directamente de fécula de maíz y de harina de trigo, mediante reacción en cadena de la polimerasa múltiple. Materiales y métodos: Se determinaron los genes toxigénicos de Bacillus cereus en muestras de ADN extraído directamente de fécula de maíz y harina de trigo, utilizando una reacción en cadena de la polimerasa múltiple específica para los genes cesB, hblC, nheA y cytK. Resultados: De 76 muestras de fécula de maíz, el 60,5% presentó los genes toxigénicos de Bacillus cereus, que fueron agrupados en seis consorcios: I: hblC, cytK (30,4%), II: nheA, hblC, cytK (21,7%), III: hblC (19,6%), IV: nheA (15,2%), V: nheA, hblC (10,9%), VI: nheA, hblC, cytK, cesB (2,2%). De 79 muestras de harina de trigo, el 65,8% presentó los genes toxigénicos de Bacillus cereus, que se agruparon en cuatro consorcios: I: nheA, hblC, cytK (80,8%), II: hblC, cytK (11,5%), III: hblC (5,8%), IV: nheA, hblC (1,9%)...

Introduction: Bacillus cereus is a human pathogen that causes two kinds of foodborne diseases, the emetic syndrome caused by emetic toxin or cereulide (Ces), and the diarrheal syndrome caused by three different enterotoxins, the hemolytic enterotoxin or hemolysin BL (Hbl), the nonhemolytic enterotoxin (Nhe) and the cytotoxin K (CytK). Objective: To determine the presence of toxigenic genes of Bacillus cereus in DNA samples directly obtained from corn starch and wheat flour using multiplex polymerase chain reaction. Material and methods: The presence of toxigenic genes of Bacillus cereus were determinedin DNA samples directly extracted from corn starch and wheat flour, using a multiplex polymerase chain reaction technique specific for cesB, hblC, nheA and cytK genes. Results: From a total of 76 corn starch samples, 60.5% had toxigenic genes of Bacillus cereus and were grouped in six consortia: I: hblC, cytK (30.4%), II: nheA, hblC, cytK (21.7%), III: hblC (19.6%), IV: nheA (15.2%), V: nheA, hblC (10.9%) and VI: nheA, hblC, cytK, cesB (2.2%). From 79 wheat flour samples tested, 65.8% had toxigenic genes of Bacillus cereus and were grouped into four consortia: I: nheA, hblC, cytK (80.8%), II: hblC, cytK (11.5%), III: hblC (5.8%) and IV: nheA, hblC (1.9%)...

Comparación de los métodos de preservación de especímenes Anopheles (Diptera: Culicidae) para la extracción de ADN / Comparison of preservation methods of Anopheles (Diptera: Culicidae) specimens for DNA extraction

Resumen Objetivos: Evaluar diferentes tratamientos de preservación de especímenes Anopheles albimanus para conocer su utilidad para conservar la cantidad y calidad del ADN que permita su análisis en estudios moleculares posteriores. Materiales y métodos: Se realizó un estudio comparativo experimental con tres bloques de tratamientos de preservación: sílica gel, etanol y congelación; los dos últimos bloques se dividieron en dos niveles cada uno: etanol absoluto, etanol al 70% y congelación a -20°C, congelación a -80°C respectivamente. El bloque control estuvo conformado por especímenes frescos sin tratamiento de preservación. Se extrajo el ADN de cada espécimen, se cuantificó por espectrofotometría y se amplifico el espaciador interno transcrito 2 (ITS2) mediante PCR. Se realizó un análisis comparativo entre bloques de tratamiento usando la frecuencia de amplificación de ITS2. Resultados: Se obtuvo la amplificación de ITS2 de todos los especímenes sin preservación previa, del 60% de los preservados en sílica gel y del 20% de los preservados en congelación a -80°C y en etanol al 70%. Se halló diferencia estadísticamente significativa (p<0,05) entre las proporciones de amplificación observadas. No se obtuvo amplificación de los especímenes preservados en congelación a -20°C y no se observó una correlación por regresión logística (p>0,05) entre el índice DO260/DO280 y la concentración de ADN de los especímenes que presentaron amplificación de ITS2. Conclusiones: Los datos sugieren que la conservación en sílica gel o la congelación que se vayan a -80°C pueden ser las mejores condiciones para la preservación de especímenes Anopheles a utilizar en estudios moleculares posteriores.

Abstract Introduction: Various methodologies have been reported for long term specimen preservation, which protect the DNA from degradation allowing its posterior analysis in molecular studies. However, the effectiveness of the preservation methods may vary among insect groups; therefore, it is convenient to determine the preservation method to be used with a particular group before performing molecular studies with a large number of specimens. Objetives: An experimental comparative study was conducted to evaluate different preservation treatments for Anopheles albimanus, to know their utility in conserving the quantity and quality of DNA to be used in subsequent molecular studies. Methods: DNA was extracted from each specimen, it was then quantified by spectrophotometer and the Internal Transcribed Spacer 2 (ITS2) was amplified by PCR. Results: ITS2 amplification was obtained in all specimens without previous preservation, in 60% of those preserved in sílica gel and in 20% of those preserved at -80°C and in 70% ethanol. A significant difference (p<0.05) was found between amplification proportions. Amplification was not obtained in specimens preserved frozen at -20°C and not correlation by logistic regression was observed between the DO260/DO280 index and DNA concentration of specimens presenting ITS2 amplification. Conclusions: The data suggest that keeping specimens in silica gel or frozen at -80°C may be the best conditions to preserve Anopheles specimens for subsequent molecular studies.

Detection of hilA gene sequences in serovars of Salmonella enterica sufigbspecies enterica

hilA gene promoter, component of the Salmonella Pathogenicity Island 1, has been found in Salmonella serovar Typhimurium, being important for the regulation of type III secretion apparatus genes. We detected hilA gene sequences in Salmonella serovars Typhi, Enteritidis, Choleraesuis, Paratyphi A and B, and Pullorum, by polymerase chain reaction (PCR) and hybridization techniques. The primers to carry out PCR were designed according to hilA sequence. A low stringency hybridization with the probe pVV441 (hilA open-reading-frame plasmid) was carried out. To find hilA gene sequences in other Salmonella sp. suggest that these serovars could have similar sequences of this kind of virulence genes

Adenoassociated virus type 2-induced inhibition of the human papillomavirus type 18 promoter in transgenic mice.

The epithelium of the cervix uteri has been reported to be frequently coinfected with both human papillomaviruses (HPV) and helper virus-dependent adenoassociated viruses (AAV). Seroepidemiological data suggest that AAV infection could inhibit cervical cancer that is caused by specific ("high-risk") types of papillomaviruses. In vitro, infection with AAV type 2 (AAV-2) or transfection of AAV-2 early (rep) genes has been shown to inhibit transformation by papillomaviruses. To analyze the effects of AAV on HPV in vivo, we studied the influence of AAV-2 infection on the promoter activity of high-risk HPV type 18 (HPV-18) in mice, transgenic for sequences of the upstream regulatory region (URR) of HPV-18 controlling transcription of the reporter gene, lacZ. Transgenic animals (or tongue cells thereof, explanted and grown in culture) were treated with dexamethasone to induce the HPV-18 promoter. Simultaneously they were (i) infected with AAV, (ii) inoculated with AAV virus-like particles (VLPs; empty capsids), or (iii) mock infected. Inoculation with AAV-2 or VLPs inhibited activation of the HPV-18 promoter. In vitro, in baby hamster kidney cells transfected with the HPV-18-lacZ construct, tissue extracts from AAV-infected animals suppressed the HPV-18 URR to a similar extent as AAV infection did. Down-regulation of the HPV-18 promoter was less efficient with extracts from animals inoculated with VLPs and was not observed with extracts from uninfected or dexamethasone-treated animals. This indicates that AAV induces cellular factor(s) in vivo capable of mediating down-regulation of the HPV-18 promoter also in cells in vitro. In contrast, promoters of the low-risk HPV types (HPV-6, HPV-11) were not influenced by AAV infection as opposed to promoters of the high-risk types (HPV-18 and HPV-16).

Detection of hilA gene sequences in serovars of Salmonella enterica subspecies enterica.

hilA gene promoter, component of the Salmonella Pathogenicity Island 1, has been found in Salmonella serovar Typhimurium, being important for the regulation of type III secretion apparatus genes. We detected hilA gene sequences in Salmonella serovars Typhi, Enteritidis, Choleraesuis, Paratyphi A and B, and Pullorum, by polymerase chain reaction (PCR) and hybridization techniques. The primers to carry out PCR were designed according to hilA sequence. A low stringency hybridization with the probe pVV441 (hilA open-reading-frame plasmid) was carried out. To find hilA gene sequences in other Salmonella sp. suggest that these serovars could have similar sequences of this kind of virulence genes.