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Ref which bacteria causes food poisoning class 8 number : Country of ref document : EP. Kind code of ref document : A2. Ref country code : DE. The invention relates to the field of multi-etiological molecular diagnosis and diagnosis of emerging diseases through the use of DNA arrays. More specifically, the invention relates to oligonucleotides included in an array for the detection of microorganisms that can cause diarrheal disease.
The array consists in immobilising said oligonucleotides in a support once they have been amplified using the PCR technique. The array can be a microarray which bacteria causes food poisoning class 8 a macroarray, and, in one embodiment, it takes the form of a biochip, either a macrochip or microchip. La presente invención pertenece a! Using DNA arrays, more specifically, the invention which bacteria causes food poisoning class 8 oiigonucleotides included in an array, for the detection of microorganisms that can cause diarrheal disease.
La O S Organización Mundial de ia Salud nos which bacteria causes food poisoning class 8 que ias enfermedades díarreicas son la mayor causa de muerte a nivel mundial en niños menores de 5 años con mas demuertes a! Actualment nos encontramos en la 7 a pandemia de cólera y esta enfermedad es endémica de varios países, siendo desgraciadamente sus mejores predictores; la pobreza, ei desplazamiento por guerras y los desastres naturales Thompson et al.
The OS World Health Organization tells us that diarrheal diseases are the leading cause of death worldwide in children under 5 with more thandeaths a! On the other hand, a large number of cases of this disease are due to the consumption of water and food contaminated by microbes viruses, bacteria and parasites. Actualment we are in the 7th cholera pandemic and the disease is endemic in several countries, unfortunately being best predictors; poverty, displacement due to wars and natural disasters Thompson et al.
The same happens in the case of infections caused by intestinal parasites causing diarrhea such as Entamoeba histoiytica, E. Ei diagnóstico de estos microorganismos se lleva a cabo de manera rutinaria en ei laboratorio a partir de un coprocutíivo, en medios de cuítívo diferenciales y de enriquecimiento, con e! The diagnosis of these microorganisms is carried out routinely in the laboratory from a coprocutivo, in differential and enrichment media, with e!
Por otra parte, se ha considerado al agente causal como un solo microorganismo, cuando es muy posible que el paciente which bacteria causes food poisoning class 8 encuentre muftí-infectado Xiroénez et al. On the other hand, There are molecular tests based on the multipiex PCR, however these tests require that the "first" be identical to the one that causes the disease having, on the one hand, many negative phases, and on the other hand, they are expensive tests that require equally expensive infrastructure PCR machine, gels, photographed eici and detect a disease while which bacteria causes food poisoning class 8 on a good previous diagnosis.
La solicitud de patente US se refiere a ia detección de genes de agentes patógenos para distinguidos de una pluralidad de no patógenos. Patent application US relates to the detection of pathogen gene genes for distinguished from a plurality of non-pathogens. La solicitud what is binary form math patente ÜS20O se refiere a oligonucteófitíos usados en microarregios para la detección de microorganismos, basado en el llamado Bacterial Digítalcode System BaDís que identifica a las bacterias por tipo, género y especie.
The patent application ÜS20O refers to oligonucleotides used in microarrays for the detection of microorganisms, based on the so-called Bacterial Digítalcode System BaDís that identifies bacteria by type, gender and species. La patente US se refiere a un método para analizar comunidades microbianas e muestras ambientales o en alimentos que consiste en el uso de microarregios en donde la intensidad de la señal es reflejo de la abundancia dei gene o de! US refers to a method for analyzing microbial communities and environmental or food samples that consists in the use of microarrays where the intensity of the signal is a reflection of the abundance of the gene or of the amount of a certain microorganism in the Sample and relative abundance data are presented.
Por consecuencia de So anterior, se ha buscado suprimir ios inconvenientes which bacteria causes food poisoning class 8 presentan los sistemas, métodos y microarregios para la detección de microorganismos del arte previo, desarrolíando un arreglo que incluye oligonucieótidos para la detección de microrganismos que pueden causar la enfermedad diarreica.
As a consequence of So anterior, it has been sought to eliminate the disadvantages of the systems, methods and microarrays for the detection of microorganisms of the prior art, developing an arrangement that includes oligonucleotides for the detection of microorganisms that can cause diarrheal disease. Tomando en cuenta las implicaciones de! Taking into account the implications of! It is an object of the present invention to provide an arrangement to quickly and accurately diagnose the potential of water and food to cause bacterial and parasitic infections causing diarrhea.
It is also another object of the present invention to provide an arrangement that can diagnose several pathogens simultaneously in both water, food and in human and animal samples. It is a further object of the present invention to provide an arrangement whose diagnosis does not give rise to false positives or negative phalluses. It is still a further object of the present invention to provide an arrangement comprising a flexible zone where environmental biological markers, which bacteria causes food poisoning class 8 Sample extraction site.
It is even more an object of the present invention to provide an arrangement for detecting the presence in a sample of Sos following enteropathogenic microorganisms: Which bacteria causes food poisoning class 8 integer aggregative whole E, coii enterotoxigenic enteric E. However, which bacteria causes food poisoning class 8 modalities, characteristics and some what is social in marketing and advantages thereof, will be better understood in the detailed description, when read in relation to the attached drawings, in which:.
La Figura 1 muestra el diseño del macroarreglo para diagnóstico de diarreas. Figure 1 shows the macroarray design for diarrhea diagnosis. Figure 2 shows the reference of organisms for the design of areas of! Figure 3 shows the design of the diagnosis by areas of the macro-region, which includes a flexible zone that can be designed for particular samples, in the case of shrimp culture, said area would include environmental biological marker genes of the sample.
La Figura 4 muestra una fotografía de una membrana expuesta a agua de mar de Ixtapa. Genes detectados: Shígelía ípaA, ipaB, ipaD, wzy Figure 4 shows a photograph of a membrane exposed to Ixtapa seawater. Genes detected: Shígelía ípaA, ipaB, ipaD, wzy La Figura 5 muestra una fotografía de una membrana expuesta a agua de mar de Puerto Vallaría. Figure 5 shows a photograph of a membrane exposed to seawater from Puerto Vallaría.
La Figura 6 muestra una fotografía de una membrana expuesta a agua de un estanque de una granja de camarones en Sinaloa. Genes de virulencia what is a linear equation in math Vibrio choferae El Tor asfB, rtxA, orfU, tcpA, Figure 6 shows a photograph of a love life quotes in one line exposed to water from a pond of a shrimp farm in Sinaloa.
Genes detected in: 16S genes of all ios colotypes, Shigella sp. The present invention belongs to the field of muitietiological which bacteria causes food poisoning class 8 diagnosis and emerging diseases, more which bacteria causes food poisoning class 8 the invention provides oiigonucleophiles useful for the defection or diagnosis of enteropathogenic microorganisms, specifically of bacteria and parasites that can cause diarrheal disease, in environmental samples, including water or food exposed to contamination or biological which bacteria causes food poisoning class 8 of animals and humans; furthermore, the invention relates to the design of arrays, including arrays, based on such oligogonucieotides of DNA to perform multiple genotyping.
In our country there is no official Mexican norm for the production and regulation of diagnostic tests based on the application of molecular or genetic technology, nor normattvity for its validation, however for the development of the present invention we have based on the reference information to the validation of diagnostic technology for the Mexican population according to the "internatíonaí Standards and Reference Materials for Quantitative Molecular Infectious Dísease Testing, ".
La presente invención provee de un macroarreglo dispuesto como macrochip para el diagnóstico de diarreas infecciosas basado en marcadores moleculares que distinguen quién es el patógeno y si es el agente etiológico. Para poder determinar sí los marcadores incluidos en la membrana son marcadores estables evolutivamente o si son sujetos a cambios debido a ía recombinación genética, se realizaron estudios evolutivos de cada uno de los marcadores en poblaciones de patógenos microbianos de todo el mundo.
Esto es relevante ya que los genes asociados a ia patogénesis pueden ser extremadamente variables debido a la selección producida por el sistema inmune, por io que tener marcadores estables del cuadro clínico es determinante para ia efectividad de ta invención. El objetivo general de! Por otra parte, debido a que en ei macroarreglo se encuentran una serie de genes que han demostrado con genética de poblaciones ser estables como unidades evolutivas, estos which bacteria causes food poisoning class 8 ser movidos por transferencia horizontal de genes y originar nuevas combinaciones que pueden generar enfermedades emergentes.
The present invention provides a macro arrangement arranged as a macrochip for the diagnosis of infectious diarrhea based on molecular markers that distinguish who the pathogen is and if it is the etiologic agent. In order to determine whether the markers included in the membrane are evolutionarily stable markers or if they are subject to changes due to genetic recombination, evolutionary studies of each of the markers were performed in populations of microbial pathogens worldwide.
This is relevant since the genes associated with the pathogenesis can be extremely variable due to the selection produced by the immune system, so having stable markers of the clinical picture is decisive for the effectiveness of the invention. The general objective of! The macro arrangement of the invention is to quickly and accurately diagnose the potential of water and food to cause bacterial and parasitic infections causing diarrhea.
The advantages offered by this method is, on the one hand, that it can simultaneously diagnose several pathogens in both water, food and human and animal samples, another advantage is that the selection of the oligo nucleotide groups of the genes listed in the tables identified with the assigned sequence number, they avoid cross hybridization between the different species, especially when they are genetically closely related and have a high level of homology; therefore, the genes selected and amplified by PCR that are included in the macro arrangement of the invention do not give rise to false positives or false negatives.
On the other hand, because in the macroarray there are a series of genes that have proven with genetics of populations to be stable as high school reunions are a waste of time units, these can be moved by horizontal gene transfer and cause new combinations that can generate emerging diseases. Finally, given its design, in the flexible zone of the macroarray it is possible to add new genes without any difficulty both to determine the ecological state of the aquatic ecosystem and to detect new diseases, both human and animal; for example, elements can be added to assess the ecosystem's ability to degrade nutrients or fix and C.
Taking advantage of the availability of data and knowledge that gives us bioinformatics, we select for the preparation of this arrangement also called maerochip, microchip or biochip virulence genes and specific genes that make up the central genome of each of the micro-organisms associated with diarrhea of which there is information, we design with them the macroarray of which bacteria causes food poisoning class 8 invention and test it; The results provide the hybridization events with the selected sequences genes corresponding to each of the enteropathogenic species.
Ya que la selección fue hecha de manera individual para cada una de las especies de manera que se garantice la detección de cepas virulentas y sin que afecte la variabilidad genética, which bacteria causes food poisoning class 8 falsos negativos, indicando la presencia o ta abundancia relativa de ias secuencias especificas presentes en ia muestra, podemos concluir que el macroarreglo de la invención es una prueba sensible, precisa y confiable, que ahorra tiempo y que es robusta, lo que permite realizar estudios de referencia epidemiológica.
Lo mismo ocurre con otros patógenos en donde en el macrochip se encuentran ios genes responsables de producir toxinas, fimbrias, factores de secreción y adherencia. El macroarregie de ia invención es una membrana de nylon, se puede utilizar cualquiera disponible en ei mercado, donde fueron impresos con un robot de alta precisión, la serie de genes amplificados previamente por PCR Figura 1. Ei macroarreglo de ia invención en su conjunto conforma un macrochip de DNA.
Since the selection was made individually for each of the species in a way that guarantees the detection of virulent strains and without affecting the genetic variability, eliminating false negatives, indicating the presence or relative abundance of the specific sequences present in In the sample, we can conclude that the macro arrangement of the invention is a sensitive, accurate and reliable test that saves time and is robust, allowing epidemiological reference studies.
For example, to detect enteropathogenic E coli EPEC Sa, which is one of the main causes of diarrhea in children under two years of age in developing countries, not only does the 16s rDNA gene tell us about the presence of E coli, but also have genes associated with the pathogenicity island LEEwhich are directly linked to the disease Sandner et al. The virulence factors on the island are the adhesion factor eae, which is one of the genes responsible for forming the secretion system type ili, escN, which excretes the adhesion gene, and two genes encoded in a plasmid, bfpB and bfpJsince both are necessary to form fimbriae that keep the bacteria which bacteria causes food poisoning class 8 to the small intestine, thus initiating the infection.
The same applies to other pathogens where the genes responsible for producing toxins, fimbriae, secretion and adhesion factors are found in the macrochip. The macroarregie of the invention is a nylon membrane, any available in the market can be used, where they were printed with a high precision robot, the series of genes previously amplified by PCR Figure 1. The macro arrangement of the invention as a whole forms a Which bacteria causes food poisoning class 8 macrochip.
La lista de genes asi como ias cepas de donde provienen fue determinada por estudios de patogénesis evolutiva y de ta literatura ver Tablas 1 y 2. The list of genes as well as the strains from which they came was determined by studies of evolutionary pathogenesis and of the what is work function and its significance see Tables 1 dirty meaning in bengali 2.
The result is a rectangular membrane with "n" known points which can be hybridized with DNA from any sample of water, feces or food. Este método es muy robusto y el hecho de que cada gen se encuentra por duplicado en la membrana what are good things to do in life evitan tanto falsos negativos como falsos positivos. Una vez realizada ía hibridización se lava la membrana para descartar todo eí ADN que no tuvo identidad.
Esta metodología puede ser ampliada a cualquier enfermedad donde se conozcan graben en la membrana ios genes responsables de la patogénesis. This method is very which bacteria causes food poisoning class 8 and the fact that each gene is duplicated in the membrane prevents both false negatives and false positives.
Once the hybridization is performed, the membrane is washed to discard all DNA that had no identity. Linear equations in two variables class 9 notes visualization is by means of chemofluorescence which can be revealed in an X-ray photographic film or by means of a cotorimometric development and is read directly on the membrane.
The pattern of points will be compared to piantillas that indicate what type of pathogen was found, what genes the sample contains and what is the potential risk! This methodology can which bacteria causes food poisoning class 8 extended to any disease where the genes responsible for pathogenesis are recorded on the membrane. Se utilizaron 35 cepas control de los diferentes patotipos de Escherichia coii y de las diferentes especies bacterianas para la amplificación por PCR de los genes de virulencia y 16S RNA que comprenden el macroarregfo.
For the design of these the following parameters were taken into account; to. Size of the initiators. Temperatura de hibridación Tm de cada uno de ios iniciadores la diferencia de la Tm entre ellos no mayor a 5X b. Hybridization temperature Tm of each of the initiators the difference of the Tm between them not greater than 5X. No formación de is self love toxic y horquillas entre ios iniciadores d.
No formation of dimers and forks between the initiators. Especificidad de ios iniciadores ai gen blanco and. Specificity of the white gene initiators. Para las amplificaciones de Sos genes por PCR, se aisló eí DNA cromosómíco de la fase estacionaria de bacterias tomadas de cultivos crecidos durante la noche. El DNA se purificó utilizando el método de miniprep descrito previamente por Murray y Thompson [1], For the amplifications of Sos genes by PCR, chromosomal DNA was isolated from the stationary phase of bacteria taken from cultures grown overnight.
Los protocolos de PCR para amplificar ios genes que conforman ia macromembrana macroarreglose realizaron de acuerdo al siguiente procedimiento con modificaciones menores en las temperaturas de hibridación.