Detection of Microsatellite Instability by High-Resolution Melting Analysis in Colorectal Cancer

Background: Colorectal cancer (CRC) is the third most common cancer worldwide. microsatellite instability (MSI) is a molecular marker of a deficient mismatch repair system and happens in almost 15% of CRCs. Because of a wide frequency of MSI+ CRC in Iran compared to other parts of the world, the importance of screening for this type of cancer is highlighted. Methods: : The most common MSI detection technique is a fluorescent PCR-based method in which fragments are analyzed by capillary electrophoresis (CE). This technique is very time-consuming, difficult, and expensive. We sought to develop and evaluate a proper method with high accuracy, specificity, and sensitivity to screen the MSI+ CRC. A high-resolution melting (HRM) analysis procedure is relying on the analysis of the melting curve attributes. Low cost, feasibility, high specificity, and sensitivity are outstanding attributes of HRM analysis. Results: Five mononucleotide microsatellite markers, including BAT-25, BAT-26, NR-21, NR-24, and NR-27, in 25 archival CRC tumor tissue samples were compared with normal tissue adjacent using HRM method. The specificity and sensitivity of BAT-25 with HRM method were 100% compared to CE, while other markers had lower sensitivity. However, when all the markers were considered together, the sensitivity and specificity became 100%. The number of MSI+ samples was 56%, which shows a higher ratio than previous Iranian studies. The highest MSI was related to BAT-26 (52%). Conclusion: The HRM method is much simpler and more cost-effective than current MSI techniques, and its sensitivity and accuracy are comparable. Therefore, it can serve as an alternative method in cases where CE is unavailable.

Diagnóstico molecular de parasitosis intestinales / Molecular diagnosis of intestinal parasitoses

Las infecciones causadas por parásitos del tracto digestivo humano representan un problema de salud pública global. En países industrializados, sus particulares características epidemiológicas (baja prevalencia general de enteroparásitos), económicas (elevados costes laborales) y clínicas (incremento constante del número de muestras y determinaciones diagnósticas a realizar) han causado que las técnicas moleculares estén progresivamente reemplazando a la microscopía convencional como método de diagnóstico de primera línea de estos patógenos en el laboratorio clínico moderno. Los métodos basados en PCR, particularmente los desarrollados para la detección simultánea de varios agentes que causan la misma etiología (diagnóstico sindrómico), representan ya una opción coste-efectiva que permite automatizar procesos, optimizar flujos de trabajo, comparar resultados entre diferentes laboratorios y facilitar la acreditación de procedimientos diagnósticos. En esta revisión se detalla de forma clara y concisa el estado actual del diagnóstico molecular de las principales especies de parásitos intestinales humanos, particularmente de los protozoos entéricos causantes de diarrea (Cryptosporidium spp., Giardia duodenalis, Entamoeba histolytica), de los miembros más destacados de los filos Microsporidia (Enterocytozoon bieneusi) y Stramenopiles (Blastocystis sp.), así como de los helmintos transmitidos por suelo (Ancylostoma spp., Ascaris lumbricoides, Necator americanus, Strongyloides stercoralis y Trichuris trichiura) y alimentos (Anisakis spp., Clonorchis sinensis, Fasciola spp., Taenia solium, y Trichinella spiralis). Especial atención se ha prestado a la descripción de técnicas y formatos disponibles, a sus prestaciones diagnósticas y a los marcadores genéticos más usados, tanto para la detección en laboratorios clínicos como para el genotipado en centros de referencia e investigación

Infections causes by parasites of the gastrointestinal tract are a global public health problem. In industrialised countries, their particular epidemiological (low general prevalence of enteroparasites), economic (high labour costs) and clinical characteristics (constant increase in the number of samples and diagnostic determinations to be performed) have led molecular techniques to progressively replace conventional microscopy as the first-line diagnostic method of these pathogens in modern clinical laboratories. PCR-based techniques, particularly those developed for the simultaneous detection of the various agents that can cause the same infectious disease (syndromic diagnosis), already represent a cost-effective option that allow process automisation, workflow optimisation, and comparison of results among different laboratories, and facilitate accreditation of diagnostic procedures. This review clearly and concisely discusses the current situation of the molecular diagnosis of the main species of intestinal parasites in humans, particularly the enteric protozoans causing diarrhoea (Cryptosporidium spp., Giardia duodenalis, Entamoeba histolytica), the most important members the Microsporidia phyla (Enterocytozoon bieneusi) and Stramenopiles phyla (Blastocystis sp.), as well as the helminths transmitted by soil (Ancylostoma spp., Ascaris lumbricoides, Necator americanus, Strongyloides stercoralis and Trichuris trichiura) and food (Anisakis spp., Clonorchis sinensis, Fasciola spp., Taenia solium, and Trichinella spiralis). Special attention is paid to the description of available techniques and formats, to their diagnostic benefits and the most widely used genetic markers for their detection, both in clinical laboratories and genotyping in referral and research centres

Comparison of TaqMan, KASP and rhAmp SNP genotyping platforms in hexaploid wheat.

Advances in high-throughput genotyping enable the generation of genome-scale data much more easily and at lower cost than ever before. However, small-scale and cost-effective high-throughput single-nucleotide polymorphism (SNP) genotyping technologies are still under development. In this study, we compared the performances of TaqMan, KASP and rhAmp SNP genotyping platforms in terms of their assay design flexibility, assay design success rate, allele call rate and quality, ease of experiment run and cost per sample. Fifty SNP markers linked to genes governing various agronomic traits of wheat were chosen to design SNP assays. Design success rates were 39/50, 49/50, and 49/50 for TaqMan, KASP, and rhAmp, respectively, and 30 SNP assays were manufactured for genotyping comparisons across the three platforms. rhAmp showed 97% of samples amplified while TaqMan and KASP showed 93% and 93.5% of amplifications, respectively. Allele call quality of rhAmp was 97%, while it was 98% for both TaqMan and KASP. rhAmp and KASP showed significantly better (p < 0.001) allele discrimination than TaqMan; however, TaqMan showed the most compact cluster. Based on the current market, rhAmp was the least expensive technology followed by KASP. In conclusion, rhAmp provides a reliable and cost-effective option for targeted genotyping and marker-assisted selection in crop genetic improvement.

An automated approach to classification of duplex assay for digital droplet PCR.

In the digital polymerase chain reaction (dPCR) detection process, discriminating positive droplets from negative ones directly affects the final concentration and is one of the most important factors affecting accuracy. Current automated classification methods usually discuss single-channel detections, whereas duplex detection experiments are less discussed. In this paper, we designed a classification method by estimating the upper limit of the negative droplets. The right tail of the negative droplets is approximated using a generalized Pareto distribution. Furthermore, our method takes fluorescence compensation in duplex assays into account. We also demonstrate the method on Bio-Rad's mutant detection dataset. Experimental results show that the method provides similar or better accuracy than other algorithms reported over a wider dynamic range.

Medical Devices; Immunology and Microbiology Devices; Classification of the Device To Detect and Identify Microbial Pathogen Nucleic Acids in Cerebrospinal Fluid. Final order.

The Food and Drug Administration (FDA or we) is classifying the device to detect and identify microbial pathogen nucleic acids in cerebrospinal fluid into class II (special controls). The special controls that will apply to the device type are identified in this order and will be part of the codified language for the device to detect and identify microbial pathogen nucleic acids in cerebrospinal fluid's classification. We are taking this action because we have determined that classifying the device into class II (special controls) will provide a reasonable assurance of safety and effectiveness of the device. We believe this action will also enhance patients' access to beneficial innovative devices, in part by reducing regulatory burdens.

Solid-phase PCR for rapid multiplex detection of Salmonella spp. at the subspecies level, with amplification efficiency comparable to conventional PCR.

Solid-phase PCR (SP-PCR) has attracted considerable interest in different research fields since it allows parallel DNA amplification on the surface of a solid substrate. However, the applications of SP-PCR have been hampered by the low efficiency of the solid-phase amplification. In order to increase the yield of the solid-phase amplification, we studied various parameters including the length, the density, as well as the annealing position of the solid support primer. A dramatic increase in the signal-to-noise (S/N) ratio was observed when increasing the length of solid support primers from 45 to 80 bp. The density of the primer on the surface was found to be important for the S/N ratio of the SP-PCR, and the optimal S/N was obtained with a density of 1.49 × 1011 molecules/mm2. In addition, the use of solid support primers with a short overhang at the 5' end would help improve the S/N ratio of the SP-PCR. With optimized conditions, SP-PCR can achieve amplification efficiency comparable to conventional PCR, with a limit of detection of 1.5 copies/µl (37.5 copies/reaction). These improvements will pave the way for wider applications of SP-PCR in various fields such as clinical diagnosis, high-throughput DNA sequencing, and single-nucleotide polymorphism analysis. Graphical abstract Schematic representation of solid-phase PCR.

Detección, identificación y localización geográfica de Begomovirus que afectan al tomate en Colombia / Detection, identification and geographical localization of tomato-infecting Begomovirus in Colombia

En la actualidad, los Begomovirus (Familia Geminiviridae) se han convertido en la mayor amenaza para los cultivos de interés agrícola ubicados en las zonas tropicales y templadas del planeta. Estos virus son transmitidos por la mosca blanca Bemisia tabaci, la cual en los últimos años en Colombia ha tenido un aumento significativo en sus poblaciones y se ha asociado con la aparición de síntomas virales en cultivos de tomate. Muestras de tomate con síntomas virales típicos fueron recolectadas en las cinco principales zonas productoras de esta solanácea en el país. Los Begomovirus fueron detectados por medio de la técnica de hibridación de ácidos nucleicos tipo Dot blot así como por medio de la reacción en cadena de la polimerasa (PCR) en todas las muestras colectadas. Con la excepción de un reporte previo en el Valle del Cauca, este es el primer reporte de Begomovirus afectando cultivos de tomate en los departamentos de Antioquia, Santander, Boyacá y Cundinamarca. Asimismo, es la primera vez que se informa sobre Begomovirus que afectan cultivos de tomate localizados por encima de 1500 msnm en Colombia.

The begomoviruses (Family Geminiviridae) have recently emerged as samples with typical Begomovirus symptoms were collected in five different departments, comprising the mayor tomato growing areas of the country. Begomovirus were detected by Polymerase Chain Reaction (PCR) or Dot Blot Hybridization in all tomato samples collected in whole tomato growing areas of the country. With exception for Valle del Cauca, this is the first report of tomato-infecting Begomovirus in Antioquia, Santander, Boyacá and Cundinamarca departments. Also this is the first report of tomato-infecting Begomovirus crops located above 1500 masl in Colombia.

Diferenciación de sinovitis transitoria y artritis séptica de cadera con criterios clínicos y ecográficos / Differentiation between transient synovitis and septic arthritis of the hip with clinical and ultrasound criteria

Introducción: La ecografía no distingue entre los diferentes tipos de sinovitis. El objetivo de este trabajo fue valorar su contribución junto a algunos datos clínicos en el diagnóstico de la artritis séptica (AS) y la sinovitis transitoria (ST) de cadera. Método: Estudio prospectivo de pacientes con ST o AS de cadera unilateral realizado entre diciembre 2006 y julio 2009. Las variables incluyeron manifestaciones clínicas y medidas ecográficas. Las ecografías fueron realizadas con un método estandarizado. Resultados: La muestra estaba compuesta por 26 niños, 22 diagnosticados de ST y 4 de AS. Se encontró diferencia en la historia de fiebre (p=0,002). La edad no era diferente, aunque la media y la mediana en ST fue 6 años, frente a media de 4,3 y mediana de 2,3 en AS. Tampoco fueron diferentes las medidas ecográficas. El valor predictivo positivo del criterio «mayor de 4 años y ausencia de fiebre» para el diagnóstico de sinovitis transitoria fue 100%, mientras que «menor de 4 años e historia de fiebre» para el diagnóstico de artritis séptica era del 75%, siempre que la radiología hubiese excluido procesos ortopédicos y la ecografía mostrara derrame. Conclusiones: Pese a las limitaciones del estudio (tamaño de la muestra y baja prevalencia) la combinación de edad e historia de fiebre parece útil para diferenciar la sinovitis transitoria de la artritis séptica. La contribución de la ecografía fue confirmar la presencia de derrame articular (AU)

Introduction: Ultrasound does not distinguish between different types of synovitis. The aim of this study was to evaluate its contribution, together with several clinical data, in the diagnosis of septic arthritis (SA) and transient synovitis (TS) of the hip. Methods: Prospective study of patients diagnosed with unilateral SA or TS of the hip carried out between December 2006 and July 2009. A set of clinical variables and ultrasound measurements were analysed. The ultrasound examinations were performed using a standardised procedure. Results: The sample included 26 children, 22 diagnosed with TS and 4 with SA. A difference was found in the history of fever (P=0.002). On the other hand, no differences were detected in the age of the children, although mean and median in the TS group were 6 years vs a mean of 4.3 with a median of 2.3 years in the SA group. There were no differences in the ultrasound measurements either. The positive predictive value of the criterion “older than 4 years of age and no history of fever” for the diagnosis of transient synovitis was 100%, while “younger than 4 years and history of fever” for the diagnosis of septic arthritis was 75%, once radiology had excluded orthopaedic processes and ultrasound showed an effusion. Conclusions: In spite of the study limitations (sample size and low prevalence) the combination of age and history of fever appears to be useful in distinguishing transient synovitis from septic arthritis. The contribution of ultrasound was to confirm the presence of joint effusion (AU)

DNA fingerprinting of Paenibacillus popilliae and Paenibacillus lentimorbus using PCR-amplified 16S-23S rDNA intergenic transcribed spacer (ITS) regions.

Failure to identify correctly the milky disease bacteria, Paenibacillus popilliae and Paenibacillus lentimorbus, has resulted in published research errors and commercial production problems. A DNA fingerprinting procedure, using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS) regions, has been shown to easily and accurately identify isolates of milky disease bacteria. Using 34 P. popilliae and 15 P. lentimorbus strains, PCR amplification of different ITS regions produced three DNA fingerprints. For P. lentimorbus phylogenic group 2 strains and for all P. popilliae strains tested, electrophoresis of amplified DNA produced a migratory pattern (i.e., ITS-PCR fingerprint) exhibiting three DNA bands. P. lentimorbus group 1 strains also produced this ITS-PCR fingerprint. However, the fingerprint was phase-shifted toward larger DNA sizes. Alignment of the respective P. popilliae and P. lentimorbus group 1 ITS DNA sequences showed extensive homology, except for a 108bp insert in all P. lentimorbus ITS regions. This insert occurred at the same location relative to the 23S rDNA and accounted for the phase-shift difference in P. lentimorbus group 1 DNA fingerprints. At present, there is no explanation for this 108bp insert. The third ITS-PCR fingerprint, produced by P. lentimorbus group 3 strains, exhibited approximately eight DNA bands. Comparison of the three fingerprints of milky disease bacteria to the ITS-PCR fingerprints of other Paenibacillus species demonstrated uniqueness. ITS-PCR fingerprinting successfully identified eight unknown isolates as milky disease bacteria. Therefore, this procedure can serve as a standard protocol to identify P. popilliae and P. lentimorbus.

Virus del papiloma humano. Aspectos virológicos e inmunitarios / Human papilloma virus. Virological and immune features

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Epidemiología de la infección por el virus del papiloma humano y del cáncer de cérvix / Epidemiology of human papilloma virus infection and cervical cancer

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Preguntas y respuestas sobre la infección por el virus del papiloma humano / Questions and answers on human papilloma virus infection

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Restos biológicos de interés forense: extracción de ADN a partir de muestras complejas / Biological remains of forensic interest: DNA extraction from complex samples

Este estudio es una breve revisión de los métodos principales para la extracción de ADN a partir de muestras complejas, como el pelo y tejidos incluidos en parafina, y como han ido evolucionando a lo largo de los años. Desde las primeras técnicas que surgieron, como la extracción orgánica, hasta los nuevos avances tecnológicos. El pelo es uno de los materiales biológicos más comúnmente asociado con las investigaciones medico legales. A pesar de ello, es una muestra delicada, y probablemente la que presente un mayor índice de fracasos a la hora de obtener resultados tras su análisis, después de los restos óseos antiguos. La extracción de ADN a partir de muestras de tejidos incluidos en parafina se realiza usualmente cuando no se dispone de muestras biológicas “frescas” del individuo implicado en una investigación forense. En estas circunstancias puede obtenerse el genotipo del individuo a partir de muestras antiguas procedentes de biopsias, cirugías o autopsias. Sin embargo, la extracción resulta complicada porque generalmente se trata de un material escaso y degradado

This study is a brief revisión of the main DNA extraction methods for complex samples, like hair and paraffin-embedded tisúes, and how they have evolved through time. We describe from the first techniques, such as organic extraction, to the new technological advances. Hair is one of the biological evidences most commonly associated to medical legal investigations. In spite of this fact, it is a delicate sample, and probably presents the larger failure ratio in order to obtain results, after skeletal remains samples. The Dna extraction from paraffin-embedded tissues samples is usually carried out when there aren´t “fresh” biological samples from an individual involved in a forensic investigation. In this ´circumstances, we can obtain the individual´s genotipe from old samples coming from biopsy, surgery or autopsy. However, the extraction is complex because generally is a poor and degraded material

Cost efficiency in the detection of chytridiomycosis using PCR assay.

Chytridiomycosis is an emerging infectious disease of amphibians associated with mass mortalities and population declines worldwide. Recent technological advances have resulted in a highly sensitive, non-invasive technique for diagnosing the disease based on a quantitative (real-time) polymerase chain reaction (qPCR) assay. The qPCR assay yields the most accurate and informative data of any available detection technique. However, due to the relatively high costs involved, it has yet to attain widespread use by chytridiomycosis researchers. Using the results of a disease survey of 467 wild frogs from eastern Queensland, Australia, we examine the necessity of triplicate assays in qPCR detection of chytridiomycosis. We describe a singlicate qPCR assay that can be used to substantially decrease costs, with no significant decrease in sensitivity. We also demonstrate that detection of chytridiomycosis by use of the conventional PCR assay may lead to appreciable underestimations in disease prevalence. We recommend that amphibian disease researchers adopt the singlicate qPCR assay as the primary means of chytridiomycosis detection.

Medical devices; immunology and microbiology devices; classification of reagents for detection of specific novel influenza A viruses. Final rule.

The Food and Drug Administration (FDA) is classifying Reagents for detection of specific novel influenza A viruses into class II (special controls). Special controls that will apply to the device are the guidance document entitled, "Class II Special Controls Guidance Document: Reagents for Detection of Specific Novel Influenza A Viruses" and limitations of distribution of these reagents. The agency is taking this action in response to a petition submitted under the Federal Food, Drug, and Cosmetic Act (the act) as amended by the Medical Device Amendments of 1976, the Safe Medical Devices Act of 1990, the Food and Drug Administration Modernization Act of 1997, and the Medical Device User Fee and Modernization Act of 2002. The agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device. Elsewhere in this issue of the Federal Register, FDA is publishing a notice of availability of a guidance document that is a special control for this device.

Desenvolvimento de uma PCR multiplex capaz de detectar e classificar cepas de Trypanosoma cruzi em amostras clínicas e de campo

Nesse trabalho, descrevemos a busca por marcadores moleculares que permitam simultaneamente o diagnóstico da doença de Chagas e a classificação das cepas segundo o fenótipo de susceptibilidade a drogas, zimodema e/ou grupos I e II do T. cruzi. [...], selecionamos seqüências repetitivas do DNA do T. cruzi e iniciadores descritos para o diagnóstico da doença de Chagas. Analisamos as seqüências do elemento repetitivo E13, kDNA e o DNA satélite (195 pb). Os resultados com o elemento repetitivo E13 e seqüências do kDNA, mostraram uma grandevariabilidade dessas seqüências, inviabilizando a busca de marcadores. Analisamos 160 seqüências do DNA satélite de 195 pb de 12 cepas do T. cruzi previamente caracterizadas segundo o fenótipo de susceptibilidade a drogas e zimodema. Estudos de filogenia mostraram a existência de dois grupos distintos, associados com os grupos I e II do T. cruzi. Observamos a presença de 8 polimorfismos exclusivos das cepas do grupo I do T. cruzi. [...] . Umsistema de PCR multiplex constituído pelos iniciadores TcSat 4 e Diaz 7 e 8 permitiram a classificação das cepas de T. cruzi nos grupos I e II. A sensibilidade foi de 10 fg, o que corresponde a 1/30 do DNA de um parasito. Amostras de DNA de outros tripanosomatídeos não produziram produto amplificado. Comparamos o perfil de amplificação do DNA satélite de 30 cepas de T. cruzi e 24 amostras de sangue de camundongos experimentalmente infectados com as cepas Colombiana (grupo I) e Y(grupo II) em papel de filtro. Em todas as amostras positivas foi possível a identificação dos grupos I e II. Para validarmos a técnica com amostras de campo, utilizamos 7 amostras de DNA do creme leucocitário de pacientes na fase aguda da doença deChagas e 15 amostras de fezes de Triatoma infestans em papel de filtro. As amostras de pacientes foram grupo II e as amostras de T. infestans grupo I. Esses resultadosestão de acordo com os dados descritos na literatura que mostram uma associação entre cepas do grupo I do T.

Desenvolvimento de uma PCR multiplex capaz de detectar e classificar cepas de Trypanosoma cruzi em amostras clínicas e de campo / Development of a PCR multiplex capable to detect and to classify cepas of Trypanosoma cruzi in clinical samples and field

Nesse trabalho, descrevemos a busca por marcadores moleculares que permitam simultaneamente o diagnóstico da doença de Chagas e a classificação das cepas segundo o fenótipo de susceptibilidade a drogas, zimodema e/ou grupos I e II do T. cruzi. [...], selecionamos seqüências repetitivas do DNA do T. cruzi e iniciadores descritos para o diagnóstico da doença de Chagas. Analisamos as seqüências do elemento repetitivo E13, kDNA e o DNA satélite (195 pb). Os resultados com o elemento repetitivo E13 e seqüências do kDNA, mostraram uma grandevariabilidade dessas seqüências, inviabilizando a busca de marcadores. Analisamos 160 seqüências do DNA satélite de 195 pb de 12 cepas do T. cruzi previamente caracterizadas segundo o fenótipo de susceptibilidade a drogas e zimodema. Estudos de filogenia mostraram a existência de dois grupos distintos, associados com os grupos I e II do T. cruzi. Observamos a presença de 8 polimorfismos exclusivos das cepas do grupo I do T. cruzi. [...] . Umsistema de PCR multiplex constituído pelos iniciadores TcSat 4 e Diaz 7 e 8 permitiram a classificação das cepas de T. cruzi nos grupos I e II. A sensibilidade foi de 10 fg, o que corresponde a 1/30 do DNA de um parasito. Amostras de DNA de outros tripanosomatídeos não produziram produto amplificado. Comparamos o perfil de amplificação do DNA satélite de 30 cepas de T. cruzi e 24 amostras de sangue de camundongos experimentalmente infectados com as cepas Colombiana (grupo I) e Y(grupo II) em papel de filtro. Em todas as amostras positivas foi possível a identificação dos grupos I e II. Para validarmos a técnica com amostras de campo, utilizamos 7 amostras de DNA do creme leucocitário de pacientes na fase aguda da doença deChagas e 15 amostras de fezes de Triatoma infestans em papel de filtro. As amostras de pacientes foram grupo II e as amostras de T. infestans grupo I. Esses resultadosestão de acordo com os dados descritos na literatura que mostram uma associação entre cepas do grupo I do T.

Localización molecular mediante PCR inverso del sitio de inserción de un transposon TN 10 ligado a gntS en mutantes de Escherichia coli / Molecular location by inverse PCR of the insertion site of a Tn10 transposón ligated to gntS in Escherichia coli mutants

La vecindad del sitio de inserción del transposón Tn10, portado por la mutante de Escherichia coli DF601, contiene el gene gntS, un presunto regulador positivo involucrado en el metabolismo del gluconato en E. coli. Aunque el análisis molecular de la región del minuto 95.3, señalado originalmente como el sitio de inserción del transposón, reveló el ORF f251 con características de regulador, transformaciones con éste y otros ORFs de la región, una vez clonados, no complementaron la función perdida en mutantes gntS. El presente trabajo racionaliza la causa de tales resultados. Con base a la secuencia nucleotídica suministrada por GenBank y la aplicación de la técnica de PCR inverso, se encontró que el sitio exacto de inserción del transposón Tn10, portado por la mencionada mutante y sus derivadas TetR, es la posición 4442377, la cual interrumpe el ORF ytfN en la región del minuto 95.8 del mapa genético y no en la del minuto 95.3, como fue originalmente establecido. Los resultados, además de señalar sin ambigüedad la región cromosómica a investigar para lograr los fines propuestos, indican la conveniencia de aplicar la técnica sencilla de PCR inverso, para ubicar elementos genéticos antes de emplearlos en estudios moleculares.

The vicinity of the Tn10 transposon insertion site, carried by the Escherichia. coli mutant DF601, contains the genegntS, a putative positive regulator involved in the metabolism of the gluconate in E. coli. Although the molecular analysis of the 95.3 minute region, originally reported as the transposon insertion site, revealed the ORF f251 as one with regulator characteristics, transformations with this and other ORFs associated with the region, once cloned, did not complement the lost function in gntS mutants. The present work rationalizes on the cause of such results. Based on the nucleotide sequence provided by GenBank and application of the inverse PCR technique, it was found that the exact site of the Tn10 transposon insertion is in the position 4442377, interrupting the ytfN ORF at the minute 95.8 of the E. coli genetic map and not at minute 95.3, as it was originally established. The results indicate the precise chromosomal region to investigate in order to obtain the initially proposed aims and the convenience of applying the simple technique of inverse PCR to locate genetic elements as well.

Molecular subtyping for Escherichia coli O157: H7 isolated in Taiwan.

Enterohaemorrhagic Escherichia coli O157: H7 is an important pathogen these days. Outbreaks of its infection have been reported all over the world, in Australia, Canada, Japan, the United States, south Africa, and various countries in Europe. In the summer of 2001, the first clinical infection by E. coli O157: H7 was identified in Taiwan. In this study, the standard procedures for molecular subtyping were applied to several strains collected in Taiwan as well as from elsewhere. The two molecular subtyping methods we used are pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). The isolates from the U.S.A., Canada, Japan, and Taiwan each showed a unique molecular fingerprinting pattern. The environmental strains isolated in Taiwan showed closer relationships with each other, and their similarity was in the range of 75-85%. The first clinical strain isolated in Taiwan in 2001 was similar to the strains from North America but not closely related to the Taiwanese environmental strains. Our surveys showed that some local E. coli O157: H7 strains did exist in Taiwan, but there had been only one official case report of the infection by local E. coli O157: H7. The eating habits of the people and the geographic distribution of the pathogen are considered crucial risk factors in Taiwan. The establishment of a database of our own and joining the global network database are important tasks if we want to control such agricultural and food-borne pathogens, and reduce the number of victims and amount sufferings, as well as the economic losses due to the infection.

Diferenciación de especies de Rhodococcus mediante una prueba de PCR-RFLP basada en los genes codificantes para la subunidad 16S ribosomal / Differentiation of Rhodococcus's species by means of a test of PCR-RFLP based on the genes codificantes for the subunit 16S ribosomal

Dada la dificultad en diferenciar las especies de Rhodococcus por pruebas bioquímicas, se desarrolló unaprueba de Reacción en Cadena de la Polimerasa (PCR), seguida de un ensayo de Polimorfismo de Longitud de Fragmentos de Restricción (PCR-RFLP) para la diferenciación de las mismas. R. equi, R. rhodnii y otrasbacterias fueron cultivadas en agar sangre y BHI a 37 y 26 °C. El ADN bacteriano fue extraído y amplificadocon los iniciadores descritos por Hypsa y Dale. Los productos de amplificación fueron sometidos a digestióncon diversas enzimas de restricción y los patrones de restricción obtenidos fueron confirmados mediante análisis in silico. Se obtuvo el fragmento de amplificación esperado de 1300 pb en todas las bacterias analizadas. Se pudo diferenciar R. equi de R. rhodnii con las endonucleasas PstI y HindIII y con respecto a otrasbacterias con PstI, HindIII, SstI, BamHI y EcoRI. Los patrones de restricción obtenidos fueron confirmadosmediante análisis in silico. La prueba PCR-RFLP constituye una alternativa para la diferenciación entreespecies de Rhodococcus tales como R. equi y R. rhodnii.