Comparative study of RFLP-IS6110 and MIRU-VNTR from Mycobacterium tuberculosis isolated in the state of Minas Gerais, Brazil

Abstract DNA genotyping of Mycobacterium tuberculosis has been widely applied in the understanding of disease transmission in many countries. The purpose of this study was to genotype the strains of M. tuberculosis isolated in patients with new tuberculosis (TB) cases in Minas Gerais, as well as to compare the similarity, discriminatory power, and agreement of the clusters between the IS6110 Restriction Fragment Length Polymorfism (RFLP) and 12 loci Variable Number Tandem Repeat - Mycobacterial Interspersed Repetitive Units (MIRU-VNTR) techniques. It was observed that 32% (66/204) of the isolated strains in the RFLP-IS6110 and 50.9% (104/204) of the isolated strains in the MIRU-VNTR presented a similarity of equal to or above 85%. The RFLP-IS6110 and MIRU-VNTR proved to contain a high discriminatory power. The similarity index resulting from the RFLP showed no recent transmission. Good agreement was observed between the techniques when clusters were detected; however, the best epidemiological relationship was found when using the RFLP-IS6110.

Diversidad genética de aislados de Phytophthora infestans colectados en zonas productoras de papa y tomate de Guatemala / Genetic diversity of Phytophthora infestans isolates collected from tomato and potato producing areas in Guatemala

Phytophthora infestans (Mont) DeBary es el agente causal de la enfermedad conocida como tizón tardío, la cual ha sido catalogada como la enfermedad de plantas más devastadora reportada en la historia de la humanidad. Este patógeno afecta plantas de importancia económica de la familia solanaceae, como el tomate y la papa. P. infestans es un oomicete heterotálico y necesita de dos tipos de apareamiento, A1 y A2, para presentar reproducción sexual, la cual es la vía por la que este patógeno incrementa su grado de diversidad, a través de una recombinación de su material genético, que representa el mayor desafío para el manejo de la enfermedad. Este estudio determinó el nivel de variabilidad genética, a través del marcador molecular amplified fragment length polymorphism (AFLP), de 22 aislados de P. infestans colectados en diferentes zonas productoras de papa y tomate. Con el perfil de bandas generado por el marcador molecular, se realizó un análisis cluster y se elaboró un dendograma de tipo unweighted pair group method with arithmetic mean (UPGMA), con el índice de Dice, mediante una matriz de distancias genéticas. Los aislados fueron situados en tres grupos principales, los cuales responden al lugar de procedencia y al tipo de planta hospedera. Se encontró un valor de similitud de 0.49 entre los aislados analizados, por lo que se concluyó que la variabilidad genética de P. infestans en Guatemala es alta.

Phytophthora infestans (Mont) DeBary is the causal agent of late blight disease, which has been cataloged as the most devasting plant disease in the history of humankind. This pathogen is capable of affecting economically important plants of the solanaceae family, such as tomato and potato. P. infestans is a heterothallic oomycete for which it needs two types of mating known as A1 and A2 to present a sexual reproduction, which is the main way by this pathogen increases its degree of genetic diversity through a recombination of its genetic material; this condition represents the major defiance to control this disease. This study determined the level of genetic variability, through the molecular marker amplified fragment length polymorphism (AFLP), of 22 P. infestans isolates collected in different potato and tomato producing areas in Guatemala. With the band profile generated by the molecular marker AFLP, a cluster analysis was performed creating a UPGMA dendrogram with Dice´s index through a genetic distances matrix. The isolates were located in three main groups, which respond to the place of origin and the type of host plant. A similarity value of 0.49 was found among the analyzed isolates. It is concluded that genetic variability of the isolates analyzed is high.

Identification of environmental mycobacteria isolated from Agra, north India by conventional & molecular approaches.

Background & objectives: Several environmental mycobacteria have been shown to be important human pathogens linked to immunomodulation especially in relation to effect on vaccination. Hence identification of mycobacteria to the species level is not only relevant to patient management but also to understand epidemiology of mycobacterial diseases and effect on vaccination. We undertook this study to assess the usefulness of various conventional and molecular methods in identification of environmental mycobacterial species from Agra, north India. Methods: One hundred nineteen isolates of environmental mycobacteria were grown from 291 (116 soil and 175 water) samples. These isolates were identified by standard biochemical tests, and a simple, rapid and cost-effective in-house developed gene amplification restriction analysis targeting 16S-23S rRNA spacer and flanking region and 16S rRNA sequencing. Results: Biochemical tests could clearly identify only 68.1 per cent (81/119) of isolates to species level. An in-house developed gene amplification - restriction analysis method could confirm the identity of 102 of 119 (85.7%) isolates and the remaining 17 isolates (14.3%) were confirmed by 16S rRNA sequencing also. These 119 environmental mycobacterial isolates, included several potentially pathogenic species such as M. fortuitum, M. chelonae, M. avium, M. marinum, M. manitobense, M. kansasii and others belonged to nonpathogenic species, M. terrae, M. smegmatis and M. flavescens. M. chelonae was isolated from water samples only whereas M. fortuitum was isolated from both water as well as soil samples. Interpretation & conclusion: The in-house developed gene amplification restriction analysis method though failed to accurately identify 14.3 per cent of isolates, facilitated rapid differentiation of most of environmental mycobacteria including potential pathogens from this area and thus would have diagnostic potential in cases with NTM infections. This combination strategy using PCR-RFLP and 16S rRNA sequencing may be useful for characterization of mycobacteria from similar environmental settings from other parts of world.

DNA polymorphism detection of Cannabis using amplified fragment length polymorphism / 法医学杂志

OBJECTIVE@#To screen the AFLP primers with good diversity to distinguish various species of Cannabis.@*METHODS@#The AFLP was used to analyze the genetic diversity of 12 species of Cannabis using 55 primer combinations.@*RESULTS@#A total of 285 AFLP bands were obtained using five primer combinations with better diversity, among which 99 bands were polymorphic and 10 bands were special, with 47-76 bands amplified in each pair of primers.@*CONCLUSION@#AFLP may has good resolution in the diversity study of Cannabis. It may provide an essential basis for further study of the genetic diversity of Cannabis.

DNA polymorphism detection of Papaver somniferum L using fluorescent amplified fragment length polymorphism / 法医学杂志

OBJECTIVE@#To detect DNA polymorphism of Papaver somniferum L using fluorescent Amplified Fragment Length Polymorphism.@*METHODS@#Genomic DNA was isolated using the AxyPrep DNA Kit, double-digested by two restrictional endonucleases (Eco RI and Mse I) and ligated to oligonucleotide adapters. After Pre-amplification and selective amplification, the DNA fragments were separated by capillary electrophoresis using the CEQ8000 DNA Fragment Analyzer.@*RESULTS@#More than 20 fragments of highly polymorphic products were obtained by 8 pairs of primer from 64 selective amplifying primer pairs.@*CONCLUSION@#The fluorescent AFLP technique can be used to detect the DNA polymorphism of Papaver somniferum.