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A total of seventeen isolates of Aspergillus flavus from maize were collected from different agro-ecological zones of Tamil Nadu, India. The isolates were tested for their ability to produce aflatoxin B1 (AFB1) in vitro by indirect competitive enzyme-linked immunosorbent assay (ELISA). The amount of AFB1 produced by the isolates of A. flavus ranged from 1.9 to 206.6 ng/ml. Among the various isolates of A. flavus, the isolate AFM46 produced the highest amount of AFB1. DNA was extracted from A. flavus isolates and their molecular variability was investigated by using restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) regions of ribosomal DNA. PCR amplification with ITS1 and ITS4 primers resulted in the amplification of a product of approximately 600 bp. Digestion of the PCR products with the restriction enzymes EcoRI, HaeIII and TaqI produced fragments of different sizes. Analysis of the genetic coefficient matrix derived from the scores of RFLP profiles showed that minimum and maximum per cent similarities among the tested A. flavus strains ranged from 0 to 88%. Cluster analysis using the unweighted pair-group method with arithmetic average (UPGMA) clearly separated the isolates into five groups (group I–V) confirming the genetic diversity among the A. flavus isolates from maize.
The development of rapid and simple typing methods is required in order to identify possible sources of human exposure to opportunistic pathogens. Klebsiella spp. belongs to a group of bacteria that are opportunistic pathogens responsible for an increasing number of multi-resistant infections in hospitals. Recently, we showed the high genetic diversity of K. oxytoca using a large collection of strains isolated from the patients of several hospitals in Poland over a 50-year period. Our results showed that the internal transcribed spacer polymerase chain reaction method (ITS-PCR) is useful for the phylogenetic delineation of genetic groups in K. oxytoca and the high discriminatory power of the PCR melting profiles (PCR MP) method can be useful for epidemiological studies of K. oxytoca. In the present study the usefulness of PCR MP was tested on two sets of strains isolated from a single unit over a short period of time. The results revealed that PCR MP has a high discriminatory power and can be useful for epidemiological studies of closely related strains of K. oxytoca isolated from a single unit over a short period of time to identify the source, reservoirs and the tract of infection spread. The advantage of PCR MP for the above application was shown by using the procedure at increasing denaturation temperature during PCR to confirm genotyping results. Considering this feature and the high discriminatory power of PCR MP, as shown in this report for determination of the genetic similarities of consecutive K. oxytoca strains, we propose that PCR MP is one of the best techniques for short-term epidemiology analysis.
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