Increasing the sensitivity of single-strand conformation polymorphism analysis of the LDLR gene mutations in brazilian patients with familial hypercholesterolemia.

Mutations in the low-density lipoprotein receptor (LDLR) gene cause familial hypercholesterolemia (FH), one of the most common single gene disorders. It is thought that FH affects approximately 1 of 500 individuals in most populations. Single-strand conformation polymorphism (SSCP) analysis is widely used to detect mutations in the LDLR gene. However, several factors such as temperature, pH, running time, gel composition and size of the DNA fragments can influence its sensitivity. We have optimized the electrophoretic conditions to screen mutations in the promoter region and exons 1-18 of the LDLR gene by varying temperature (5 degrees C, 8 degrees C, 12 degrees C and 15 degrees C), voltage (300 to 600 V), and running time (1 to 4 hours) in the semi-automated GenePhor system (Amersham Biosciences). The efficiency of the method was evaluated by using 30 positive controls (DNA samples with mutations and polymorphisms in the LDLR gene, previously characterized) and DNA samples from 90 Brazilian patients with FH. Our results show that the use of two temperatures (5 degrees C and 15 degrees C) in combination with other optimized conditions resulted in high mutation detection rate (97%), which was considered appropriate for routine screening. Therefore, this strategy could be useful for the diagnosis of genetic disorders, cancer, and for pharmacogenetic studies.

Standardization of denaturing gradient gel electrophoresis for mutant screening of influenza A (H3N2) virus samples.

Because of the extensive genetic variability of the influenza viruses, new virus mutants arise worldwide. In the human population, some strains may become potentially epidemic after evading the immune response of the host. At present, molecular methods have made it possible to identify these variants. However, if a large number of samples need to be analyzed the identification of randomly mutated nucleotides cannot be achieved by sequencing analysis or restriction fragment length polymorphism (RFLP). In order to improve this process, a denaturing gradient gel electrophoresis (DGGE) protocol capable of discriminating between reference strains representative of different influenza seasons, some mutant strains, and five clinical isolates was standardized Ribonudeic acid (RNA) was isolated and submitted to a one-step RT-PCR that amplified the region codifying for the globular domain of the Haemagglutinin (HA) molecule. The amplicons were analyzed by electrophoresis in 6% polyacrylamide gel at 60 degreeC/150 V for 8 h, using a 31--41% urea--formamide gradient. This method was able to distinguish between closely related nucleotide sequences, confirming its suitability as screening methodology for the analysis of influenza virus epidemiology, by allowing a faster and more extensive evaluation of a large number of the variant strains detected in a specific region of the world.

Standardization of procedures of Plasmodium falciparum antigen preparation for serologic tests.

The objective of the present study is to standardize the technical variables for preparation and storage of Plasmodium falciparum and of antigen components extracted with the amphoteric detergent Zwittergent. P. falciparum obtained from in vitro culture was stored at different temperatures and for different periods of time. For each variable, antigen components of the parasite were extracted in the presence or absence of protease inhibitors and submitted or not to later dialysis. Products were stored for 15, 30 and 60 days at different temperatures and immunological activity of each extract was determined by SDS-PAGE and ELISA using positive or negative standard sera for the presence of IgG directed to blood stage antigens of P. falciparum. Antigen extracts obtained from parasites stored at -20 degrees C up to 10 days or at -70 degrees C for 2 months presented the best results, showing well-defined bands on SDS-PAGE and Western blots and presenting absorbance values in ELISA that permitted safe differentiation between positive and negative sera.

Variación temporal de electroferotipos y serotipos de Rotavirus humanos en el noroeste argentino / Temporal variation of human rotavirus electropherotypes and serotypes in the northwest of Argentine

La epidemiología de los Rotavirus se investigó en Tucumán entre los años 1986 y 1991. Durante este período se estudiaron 1462 muestras fecales de niños con gastroenteritis, de edades comprendidas entre los 0 y 5 años. La detección viral se realizó por ELISA específico para Rotavirus del grupo A y electroforesis del ARN viral en gel de poliacrilamida (PAGE). Resultaron positivas 353 muestras (24,1 por ciento). El análisis de los electroferotipos (EFT) permitió establecer la prevalencia de cepas circulantes a patrón largo y corto, la permanencia de lagunas en años sucesivos, desaparición y presencia de nuevos EFT. Se detectaron brotes periódicos de EFT cortos en los años 1987 y 1990. Exceptuando un EFT, todos fueron característicos del grupo A de Rotavirus. Del total de muestras positivas, 127 fueron serotipificadas mediante técnica de ELISA, con anticuerpos monoclonales, dirigidos contra VP7, polipéptido principal de la cápside externa viral, con especificidad para los serotipos 1, 2, 3 y 4. Los resultados mostraron una predominancia variable en diferentes años. Los datos presentados sugieren: 1)cambios permanentes de los subpatrones electroforéticos; 2)variación cíclica de los serotipos en los 6 años estudiados

Variación temporal de electroferotipos y serotipos de Rotavirus humanos en el noroeste argentino / Temporal variation of human rotavirus electropherotypes and serotypes in the northwest of Argentine

La epidemiología de los Rotavirus se investigó en Tucumán entre los años 1986 y 1991. Durante este período se estudiaron 1462 muestras fecales de niños con gastroenteritis, de edades comprendidas entre los 0 y 5 años. La detección viral se realizó por ELISA específico para Rotavirus del grupo A y electroforesis del ARN viral en gel de poliacrilamida (PAGE). Resultaron positivas 353 muestras (24,1 por ciento). El análisis de los electroferotipos (EFT) permitió establecer la prevalencia de cepas circulantes a patrón largo y corto, la permanencia de lagunas en años sucesivos, desaparición y presencia de nuevos EFT. Se detectaron brotes periódicos de EFT cortos en los años 1987 y 1990. Exceptuando un EFT, todos fueron característicos del grupo A de Rotavirus. Del total de muestras positivas, 127 fueron serotipificadas mediante técnica de ELISA, con anticuerpos monoclonales, dirigidos contra VP7, polipéptido principal de la cápside externa viral, con especificidad para los serotipos 1, 2, 3 y 4. Los resultados mostraron una predominancia variable en diferentes años. Los datos presentados sugieren: 1)cambios permanentes de los subpatrones electroforéticos; 2)variación cíclica de los serotipos en los 6 años estudiados (AU)