Portable diagnostic platform for detection of microorganisms Coliforms and E. coli.

Portable diagnostic devices are a viable and low-cost alternative for the detection of pathogens, since they reduce the time of analysis of results availability. Ease of sample collection and quick diagnosis allow this new input to be applied in the diagnosis of the main contaminating microorganisms present in the water. Laboratory tests evaluated the technical viability of the diagnostic device, using commercial strains which were inoculated and optimized in the devices and their growth compared to the conventional method in Petri dishes. Samples of 100 μL bacterial suspension were tested and compared with the traditional sample inoculation method. The device viability was determined by detecting characteristic bacterial colonies in a specific culture medium through the colorimetric development of the obtained colonies. The feasibility assessments allow us to affirm that the device enables both qualitative and quantitative detection of the target bacteria present in liquid samples, and is promising to be applied to assess the quality of water, food and environmental surfaces

Transcriptome Analysis of the Effects of Gomisin A on the Recovery of Carbon Tetrachloride-Induced Damage in Rat Liver

Gomisin A possesses a hepatic function-facilitating property in liver-injured rats. Its preventive action on carbon tetrachloride-induced cholestasis is due to maintenance of the function of the bile acids-independent fraction. To investigate alterations in gene expression after gomisin A treatment on injured rat liver, DNA microarray analyses were performed on a Rat 44K 4-Plex Gene Expression platform with duplicated reactions after gomisin A treatment. We identified 255 up-regulated and 230 down-regulated genes due to the effects of gomisin A on recovery of carbon tetrachloride-induced rat liver damage. For functional characterization of these genes, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes biochemical pathways analyses were performed. Many up-regulated or down-regulated genes were related to cell cycle or focal adhesion and cell death genes, respectively. Our microarray experiment indicated that the liver repair mechanism induced by gomisin A was strongly associated with increased gene expressions related to cell cycle and suppression of the gene expression related in cell death

Identificação de alvos moleculares para o diagnóstico quantitativo da resistência a pesticidas organofosforados em populações do carrapato-dos-bovinos.

Os programas de controle direcionados aos parasitas ainda exigem a utilização de produtos químicos para maior estabilidade operacional das ações de controle e o uso de carrapaticidas químicos é a principal ferramenta de controle para as infestações do carrapato-dos-bovinos. A utilização de técnicas fenotípicas de avaliação in vitro da suscetibilidade de carrapatos às bases pesticidas são ainda o método mais prático e mais utilizados para o diagnóstico e mensuração do fator de resistência às bases carrapaticidas. Na atualidade, o desenvolvimento e o aprimoramento de provas diagnósticas moleculares de resistência aos diferentes grupos pesticidas devem ser consideradas como ações prioritárias de pesquisa e desenvolvimento dada a necessidade de disponibilização ao setor produtivo de ferramentas acuradas e de rápida execução que a médio prazo venham a substituir os bioensaios, os quais necessitam de aproximadamente 45 dias para obtenção dos resultados. A partir de uma população de Rhipicephalus microplus reconhecidamente resistente a pesticidas foram realizados os estudos que demonstram a atividade enzimática das estares no processo de detoxicação de organofosforados. Observou-se que na cepa de R. microplus resistente a organofosforados ocorreu o aumento na transcrição dos genes que codificam enzimas metabolizantes, possibilitando identificar os alvos moleculares aptos para serem utilizadas no desenvolvimento do ensaio diagnóstico molecular quantitativo para a resistência aos pesticidas organofosforados em populações do carrapato-dos-bovinos.bitstream/item/139148/1/bpd-74-carrapatodosbovinos.pd

Bioanalytical of alicyclobacillus acidoterrestris : detection in citric fruits, isolation microbiology and phylogenetic classification by biomolecular techniques and microchips electrophoresis\"

Neste trabalho desenvolvemos métodos analíticos e moleculares para a detecção, isolamento e classificação filogenética de Alicyclobacillus spp. a partir de sucos de laranja e frutos ácidos, pelas técnicas: RT-PCR, nested RT-PCR, RAPD, seqüenciamento do 16S rRNA e análise por métodos eletroforéticos. A sensibilidade na detecção dos A. acidoterrestris foi melhorada por meio de reações de nested RT-PCR, utilizando primers internos (amplicon de 191 bp) que foram desenhados a partir do primeiro amplicom de 294 bp. O limite de detecção foi estudado com as reações RT-PCR e nested RT-PCR, sendo capazes de detectar concentrações de 0,1 UFC mL-1 para culturas puras e 2 UFC mL-1 em sucos de laranja artificialmente inoculados. A inibição de esporos de A. acidoterrestris também foi estudada para monitorar a diminuição da viabilidade com tratamento térmico e Sapindus saponaria (200 mg L-1), utilizando RT-PCR e nested RT-PCR. Com o tratamento térmico de 99 oC por 1 h o grau de inibição dos esporos foi de 96,3%. Enquanto que, com a fração purificada de S. saponaria (200 mg L-1) incubada à 45 oC por 2 dias foi de 93,6%, mas com incubação de 99 oC por 1 h foi de 98,7%, na mesma concentração de saponina. Todas as análises de quantificação de produtos de RT-PCR e nested RT-PCR foram analisadas por meio de eletroforese em gel de agarose e eletroforese capilar em microchip no Bioanalyzer 2100 (Agilent), com os kits DNA 500 e DNA 1000 LabChip®, obtendo-se maior sensibilidade nos microchips. A classificação molecular de dezenove cepas, isoladas a partir de diferentes sucos e frutos ácidos, foram estudadas utilizando RAPD-PCR e eletroforese capilar em microchips. Utilizando cinco primers aleatórios nas reações de RAPD, foi possível estudar os polimorfismos analisados nos microchips. Segundo as análises eletroforéticas, as cepas de suco concentrado e diluído de laranja (1, 2, 6) suco concentrado de limão (lim) e suco de laranja in natura (T2, T3), apresentaram similaridades genéticas com a A. acidoterrestris. O estudo de análise filogenética baseada na comparação de seqüências de DNA da região variável do gene 16S rRNA de Alicyclobacillus acidoterrestris, foi utilizado para identificar e agrupar onze cepas isoladas de superfícies e sucos de frutos ácidos. Na árvore filogenética gerada pelo método neighbor joining e bootstrap 1000x, as cepas analisadas mostraram similaridades de 99% entre todas elas, observando-se uma maior similaridade do controle A. acidoterretris (C2) com a cepa isolada de suco concentrado de limão (lim), e uma boa discriminação entre controles das espécies A. acidocaldarius, A. cicloheptanicus, A. sendaiensis e Sulfobacillus acidophilus.In this work, we developed analytical and molecular methods for detection, isolation and phylogenetic classification of Alicyclobacillus spp. from orange juice and acid fruits using RT-PCR, nested RT-PCR, RAPD and sequencing of 16S rRNA techniques and electrophoretic methods of analysis. The sensitivity on the detection of A. acidoterrestris in orange juice was improved by nested RT-PCR, using internal primers (amplicon of 191 bp) that were designed after sequencing the first amplicon (294 bp). The detection limit was studied with RT-PCR and nested RT-PCR assay, it was able to detect concentrations of 0.1 UFC mL-1 for media culture and 2 UFC mL-1 in inoculated orange juice. The inhibition in spores from A. acidoterrestris was also studied to monitoring the diminution of viability with heat treatment and Sapindus saponaria (200 mg mL-1), using RT-PCR and nested RT-PCR assays. The inhibition by heat treatment at 99 oC for 1 h was 96.3%. However, incubation with S. saponaria at 45 oC for 2 days inhibited 93,6%, however, with incubation of 99 oC for 1 h was 98,7%, in the same concentration of saponin. The quantification of the RT-PCR and nested RT-PCR amplification product were accomplished by capillary electrophoresis in microchips using the Bioanalyzer 2100 in conjunction with the LabChip (TM) DNA 500 and DNA 1000. The molecular classification of nineteen strains isolated from different juice and acidic fruit, were studied using RAPD-PCR and capillary electrophoresis in microchips. Using five random primers in the RAPD assay, it was possible to study the polymorphisms analyzed in microchips. According to electrophoresis analyses, the strains from concentrated and diluted orange juices (1, 2, 6), lemon concentrated juice (lim) and natural orange juice (T2, T3), showed genetic similarities with the A. acidoterrestris. The study of the phylogenetic analyses based on DNA comparison sequences of the variable region of 16S rRNA gene from Alicyclobacillus acidoterrestris, was utilized for the identification and grouping of eleven strains isolated from surface and juice of acid fruits. In the phylogenetic tree produced by neighbor joining and bootstrap 1000, the strains showed similarities of 99% among all strains, showing a high similarity of A. acidoterrestris with a strain isolated from lemon concentrate juice (lim), and a good discrimination between the species A. acidocaldarius, A. cycloheptanicus, A. sendaiensis and Sulfobacillus acidophilus

A Comparison of Plating and Reverse Transcriptase Polymerase Chain Reaction Followed by Microchip Electrophoresis for the Inactivation of Alicyclobacillus acidoterrestris Using Saponin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)It was used reverse-transcriptase polymerase chain reaction (RT-PCR) followed by capillary electrophoresis on a microchip to probe the viability of Alicyclobacillus acidoterrestris spores after inactivation with saponin and heat. The method was shown to be suitable for the purpose, and was faster and more sensitive than the traditional plating technique, the standard of the food industry. The limits of quantification and detection were 0.0107 and 0.0039 ng mu L-1 of amplified DNA, respectively. The correlation coefficient (r) between traditional plating and our method was 0.9977, which indicates an excellent correspondence between them. Consequently, it was possible to assess, with confidence, the viability of bacteria using the RT-PCR reaction for detection. It was evaluated the potential of this molecular method over traditional microbiology in the inactivation of A. acidoterrestris by saponin as an effective agent, potentiated by heat.2519197Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)INCTBioConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2001/14391-4

A Comparison Of Plating And Reverse Transcriptase Polymerase Chain Reaction Followed By Microchip Electrophoresis For The Inactivation Of Alicyclobacillus Acidoterrestris Using Saponin

It was used reverse-transcriptase polymerase chain reaction (RT-PCR) followed by capillary electrophoresis on a microchip to probe the viability of Alicyclobacillus acidoterrestris spores after inactivation with saponin and heat. The method was shown to be suitable for the purpose, and was faster and more sensitive than the traditional plating technique, the standard of the food industry. The limits of quantification and detection were 0.0107 and 0.0039 ng μ-1 of amplified DNA, respectively. The correlation coefficient (r) between traditional plating and our method was 0.9977, which indicates an excellent correspondence between them. Consequently, it was possible to assess, with confidence, the viability of bacteria using the RT-PCR reaction for detection. It was evaluated the potential of this molecular method over traditional microbiology in the inactivation of A. acidoterrestris by saponin as an effective agent, potentiated by heat. © 2014 Sociedade Brasileira de Química.2519197Eguchi, S.Y., Manfio, G.P., Pinhatti, M.E.M.C., Azuma, E., Variane, S.F., (1999) Acidothermophilic Sporeforming Bacteria (ATSB) in Orange Juices: Detection Methods, Ecology and Involvement in the Deterioration of Fruit Juices, 1. , 2nd ed.Fundação André Tosello & ABECitrus: CampinasEiroa, M., Junqueira, V., Schmidt, F., (1999) J. Food Protect., 62, p. 883Pettipher, G.L., Osmundson, M.E., Murphy, J.M., (1997) Lett. Appl. Microbiol., 24, p. 185Splittstoesser, D.F., Churey, J., Lee, C.Y., (1994) J. Food Protect., 57, p. 1080Yamazaki, K., Tekuda, H., Shinano, H., (1996) Biosci. Biotechnol. Biochem., 60, p. 543Orr, R.V., Shewefelt, R., Huang, C., Tefera, S., Beuchat, R.L., (2000) J. Food Protect., 63, p. 1517Knorr, D., (1995) New Methods of Food Preservation, 1. , 2nd ed.Blackie: LondonButz, P., (2002) Food Res. Internat., 35, p. 279Komitopoulou, E., Boziaris, I.S., Davies, E.A., Delves-Broughton, J., Radstrom, M.P., (1999) Int. J. Food Science Technol., 34, p. 81Yamazaki, K., Murakami, M., Kawai, Y., Inoue, N., Matsuda, T., (2000) Food Microbiology, 17, p. 315Grande, M.J., Lucas, R., Abriouel, H., Omar, N.B., Maqueda, M., Martínez-Bueno, M., Martínez-Cañamero, M., Gálvez, A., (2005) Int. J. Food Microbiology, 104, p. 289Minamikawa, M., Kawai, Y., Inoue, N., Yamazaki, K., (2005) Curr. Microbiology, 1, p. 22Falcone, P.M., Campaniello, D., Altieri, C., Sinigaglia, M., Corbo, M.R., Anese, M., Del Nobile, M.A., (2005) Ital. J. Food Sci., p. 142. , special issueBevilacqua, A., Corbo, M.R., Sinigaglia, M., (2008) Int. J. Food Sci. Technol., 43, p. 1271Hostettmann, K., Marston, A., (1995) Chemistry and Pharmacology of Natural Products, 1. , 3rd ed.Cambridge University Press: CambridgeFrancis, G., Kerem, Z., Makkar, H.P.S., Becker, K., (2002) Brit. J. Nutrit., 88, p. 587Sparg, S.G., Light, M.E., Van Staden, J., (2004) J. Ethnopharmacol., 94, p. 219Tsuzuki, J.K., Svidzinski, T.I.E., Shinobu, C.S., Silva, L.F.A., Rodrigues-Filho, E., Cortez, D.A.G., Ferreira, I.C.P., (2007) Anais Acad. Bras. Cienc., 79, p. 577Sheridan, G.E., Masters, C.I., Shallcross, J.A., MacKey, B.M., (1998) Appl. Environ. Microbiol., 64, p. 1313Williams, J.M., Trope, M., Caplan, D.J., Shugars, D.C., (2006) J. Endod., 32, p. 715McKillip, J.L., Jaykus, L.A., Drake, M., (1998) Appl. Environ. Microbiol., 12, p. 4264Nachamkin, I., Panaro, N., Huong Ung, M.L., Kricka, L., Wilding, P.J., (2001) J. Clin. Microbiol., 39, p. 754Gottwald, E., Müller, O., Polten, A., (2001) Electrophoresis, 22, p. 4016Jassona, V., Jacxsens, L., Luning, P., Rajkovic, A., Uyttendaele, M., (2010) Food Microbiol., 27, p. 710Yamazaki, K., Tekuda, H., Inoue, N., Shinano, H., (1996) Letters in Appl. Microbiology, 2, p. 350Alberice, J.V., Funes-Huacca, M.E., Guterres, S.B., Carrilho, E., (2012) Int. J. Food Microbioogy., 159, p. 130Panaro, N.J., Yuen, P.K., Sakazume, T., Fortina, P., Kricka, L.J., Wilding, P., (2000) Clin. Chem., 11, p. 1850Jabasini, M., Zhang, L., Dang, F., Xu, F., Almofli, M.R., Ewis, A.A., Lee, J., Baba, Y., (2002) Electrophoresis, 23, p. 1537Sodowich, I., Fadl, I., Burns, C., (2007) Electrophoresis, 28, p. 236