Salmonellae are enteric bacteria infecting animals and humans. Large animal clinics and Veterinary Teaching Hospitals are greatly affected by Salmonella outbreaks and nosocomial infection. The risk of environmental contamination and spread of infection is increased when animals are confined in close contact with each other and subjected to increased stress factors. This study was designed to compare double-enrichment culture techniques with Gel-based and Real-time PCR assays in the quest for improved diagnostic methods for detecting Salmonella in equine fecal samples. 120 fecal samples submitted to the Clinical Microbiology Laboratory of the Veterinary Medical Teaching Hospital at Texas A&M University (CML, VMTH, TAMU) were tested for Salmonella using all three techniques. Double-enrichment bacterial culture detected 29 positive results (24%), Real-time PCR detected 33 positive results (27.5%), and Gel-based PCR detected 73 positives results (60.8%). While culture and real-time PCR methods had similar results, the gel-based PCR method detected many more positive results, indicating probable amplicon contamination. Real-time PCR can be completed as soon as the day after submission while culture techniques may take 2 to 5 days to complete. However, viable bacterial cells are needed for antimicrobial susceptibility testing and serotyping: both important for epidemiological studies. Therefore, double-enrichment bacterial culture performed concurrently with real-time PCR methods could be efficient in clinical settings where both accurate and expedient results are required.

No description available.

In order to determine the effects that Maillard Reaction Products (MRP) have on Salmonella Typhimurium, growth rates and virulence expression, in the presence of Maillard reaction products, were observed, using the β−galactosidase Miller Assay and Reverse Transcription Polymerase Chain Reaction (RT-PCR). The presence of MRP compounds in liquid media caused no negative effect on the growth rate of Salmonella cells. However, the addition of MRP compounds at a 1% level in the media caused a significant increase in hilA expression in Salmonella Typhimurium, and the highest induction levels were observed in media supplemented with arginine and histidine-MRP compounds. There was no effect on the induction of hilA with the 0.5% addition of the MRP compounds in the amended media as shown by the Miller Assay. However, there was an effect seen when using the Real Time RT-PCR assay that resulted in the same levels of significance seen at 1.0% additions of MRPs being seen at the 0.5% level as well. Since rsmC was shown to be a constitutive gene that had continuous levels of expression in Salmonella based on cell number, Real-Time PCR was then used to assess the hilA expression of Salmonella Typhimurium under different oxygen, pH levels, and osmolarity conditions. The results under low oxygen indicate that the combination of low osmolarity and high pH have the highest inducing effect on hilA expression. The hilA response under the same media conditions and a high oxygen environment showed the same pattern of expression as those bacteria grown under a non-aerobic environment. The media with a pH of 8 and low osmolarity conditions had the greatest effect on the induction of hilA with none of the other media showing any significant effect. The relative expression of hilA did decrease for those bacteria grown under aerobic conditions versus those grown under low oxygen conditions.

This report is the result of a project in the course Independent Projekt in Molecular Biotechnology at Uppsala University during the spring of 2014. The foremost purpose of the course is to give students the opportunity to carry through exstensive work in a project environment. This project was formed based on a comission from the biotechnology company SweTree Technologies, and the goal has been to compose a summary of the different techniques and methods that exist in the field of mass propagation of trees through the method of somatic embryogenesis. The project group has obtained information about the area mainly throgh reading patents, trying to find key components and bottlenecks in other companies’ somatic embryogenesis technologies. This paper is divided into different sections, containing the patents of the automation of different steps in the process. This is to make it easier for readers to find information about the area they are interested in, as well as to illustrate the main parts of the process as percieved by the project group. Currently, there are several automated solutions for almost every step in the process, some of which are already in use. All the information obtained shows that the cost and labour has decreased with the development of this technology. While there is still room for significant devolopment in order to produce a complete automated process, there is no doubt that this method is becoming an ever more important asset in the area of forestry. Our hope is that this report may be a useful tool for companies or laymen to geta grasp of the field of automated mass production of trees.

In 1995 and again in 1998, millions of pilchards (Sardinops sagax neopilchardus) were found dead or dying off the coast of Australia and also in New Zealand. The epizootics moved progressively, at a rapid speed against the prevailing currents. A previously unrecognised herpesvirus, Pilchard herpesvirus (PHV), was identified as the causative agent. Until recently, rapid and sensitive methods to detect PHV were not available and based on a previously identified and conserved 373 bp region of the genome, polymerase chain reaction (PCR), in situ hybridisation and real-time PCR methods were developed for the specific detection of PHV in formaldehyde-fixed and frozen tissues of pilchards. Real-time PCR was shown to have greater sensitivity than a conventional PCR and in situ hybridisation for the detection of PHV infection. The PCR assay and sequence analysis of the amplification products was used to compare the 373 bp region of the genome from strains obtained during the 1995 and 1998 epidemics. Significant differences between the strains were not detected. Additional sequence data was obtained adjacent to the 373 bp of known PHV sequence, which did not match any sequence in any of the genetic databases, and this will be invaluable for further study of the pilchard herpesvirus and the development of improved detection methods. The molecular-based methods of virus detection developed were applied to a reexamination of virus in paraffin-embedded tissues taken from fish during an attempt to transmit the virus to wild caught pilchards in 1999. The results obtained confirmed previously equivocal results that transmission of PHV to wild caught pilchards was achieved, although this experiment failed to demonstrate that transmission of the virus resulted in severe lesions typical of those seen in the epizootics. Using formaldehyde-fixed samples from fish collected during the 1998 PHV epizootic, virus was detected in fish collected 4 days prior to the occurrence of the epizootic even though the fish then appeared clinically normal, during the epizootic, and 8 days after mortalities had ceased. An investigation of wild pilchards collected from 4 Australian pilchard subpopulations using real-time PCR demonstrated that PHV was present in the gills of 13.75% of 800 fish sampled, indicating that the virus is now endemic in the Australian pilchard population. Variation in the prevalence of PHV infection in the 4 subpopulations was detected, higher in western and southern populations than in populations from the east coast. The endemic nature of PHV infection in the pilchard population explains why there have been no further epizootics with mass mortalities since 1998.

Ulm, Univ., Diss., 2008.

Objective: Describe the prevalence of HPV genotypes via PCR and DNA sequencing in 397 women who attended to the gynecological outpatient center in the Hospital Regional Docente de Cajamarca from March to September 2017. Results: A positive PCR result for HPV was observed in 121 cervical samples. A high-risk genotype was found in 63.6% (77/121) of patients, a probably oncogenic type in 23.1% (28/121) and a low-risk type in 7.4%. Among the high-risk genotypes, HPV-31 was the most common one present in 20% (21/77), followed by HPV-16 in 11.4% (12/77). Coinfections between two or more genotypes were observed in 12 cases. / This work was supported by 4th research incentive of the Universidad Peruana de Ciencias Aplicadas (Grant: UPC‑EXP‑02‑2017). Lima, Peru. / Revisión por pares

El presente trabajo de título tiene por objetivo la detección de árqueas en procesos de biolixiviación, y el estudio de la relación que pudiese existir entre la variación de los parámetros fisicoquímicos presentes en el ambiente en estudio y la presencia de árqueas para cada condición estudiada. Utilizando una mini columna rellena con mineral sulfurado y alimentada como media basal conteniendo sulfato ferroso, a la que se hicieron una serie de cambios, como la temperatura dentro de ésta y los iones presentes en las soluciones utilizadas. Las temperaturas utilizadas para este estudio fueron de 45 °, 55° y 65° C. Para lo anterior, se realizó el análisis mediante reacción de PCR de las árqueas presentes en las soluciones obtenidas de una mini columna de biolixiviación montada en el Laboratorio de Biohidrometalurgia. La ventaja de que presenta este tipo de análisis es el ser independiente de cultivos, por lo que permite obtener una aproximación de los organismos presentes en solución, en particular en el caso de árqueas, microorganismos difíciles de cultivar. La técnica utilizada consiste en realizar la concentración de las células presentes en la solución, la extracción del DNA de las células, la reacción de PCR con primers universales específicos para árqueas en procesos de biolixiviación y la posterior visualización en gel de agarosa del producto de PCR. Para realizar el PCR, se seleccionó un par de primers universales para árqueas desde la literatura, realizando un PCR virtual, mediante programas computacionales y analizando los productos amplificados teóricos; el criterio de obtener los productos más ho,homogéneos en cuando a tamaño y representatividad respecto a la muestras.

A Doença de Marek é uma enfermidade linfoproliferativa das aves, causada por um alfaherpesvírus e caracterizada pela infiltração de células em nervos periféricos, gônadas, íris, vísceras, músculos e pele. Desde 1970, vacinas atenuadas têm sido utilizadas como ferramenta principal no controle da doença. Esse trabalho descreve a implantação da Reação em Cadeia da Polimerase em Tempo Real (qPCR) para a titulação de vacinas contra o vírus da doença de Marek do sorotipo 3, herpesvírus dos perus (HVT). A qPCR foi comparada com a técnica tradicional de titulação, baseada no cultivo celular de fibroblastos de embrião de galinha. Foram avaliadas três vacinas vivas (congeladas, cepa FC126) provenientes de distintos fabricantes. A técnica molecular apresentou alta correlação entre os valores de threshold cycle (CT) e respectivas diluições das vacinas (R2 = 0,99), indicando que, dentro desta faixa linear testada (102 a 104 PFU/dose), a qPCR foi capaz de quantificar as vacinas disponíveis no mercado. A reprodutibilidade da titulação em cultivo celular e qPCR foi avaliada pela realização dos testes em três dias distintos a partir de ampolas de um mesmo lote da vacina. Os títulos obtidos por ambos os métodos demonstraram alta reprodutibilidade e coerência com o fornecido pelo fabricante. Caracterizou-se também a proporção de vírus livres e associados às células, onde foi observado que, pelo menos, 90% dos vírus encontravamse na forma associada. Este trabalho indicou que a qPCR é reprodutível, rápida e menos trabalhosa do que a titulação em cultivo celular tradicionalmente utilizada.

This thesis deals with possibilities of isolation DNA yeasts genus Brettanomyces from wine with following identification using PCR. Fourteen wine samples with assumption of occurrence Brettanomyces were analysed. The literary section describes characteristic of yeasts Brettanomyces and their impact on wine. Further is described principle of PCR and use of this method in detection of Brettanomyces. Four different methods of isolation DNA directly from wine were tested. Wine samples were analysed using of five different primers by PCR. In this work were tested designed probe and primers for identification of Brettanomyces by real-time PCR.