Molecular Differentiation of Selected <i>Pandanus</i> spp. by Random Amplified Polymorphic DNA (RAPD) Analysis

Clinical isolates of Microsporum canis and M. gypseum from humans, dogs and cats were examined by random amplification of polymorphic DNA (RAPD) and Southern hybridization analyses. The RAPD band patterns of six clinical isolates of M. canis were identical to those of standard strains of Arthroderma otae. Of nine clinical isolates of M. gypseum seven and two isolates showed RAPD patterns identical to those of standard strains of A. gypseum and A. incurvatum respectively. Southern blot analysis using a probe (C3) obtained from A. otae DNA revealed that six clinical isolates of M. canis showed specific bands identical to those detected in the standard strains of A. otae. Of nine clinical isolates of M. gypseum, seven and two isolates showed bands hybridized by the C3 probe identical to those detected in A. gypseum and A. incurvatum respectively. Furthermore, the results from mating experiments on these nine clinical isolates of M. gypseum showed complete agreement with the results from RAPD and Southern hybridization analyses. These findings clearly indicate that RAPD and Southern hybridization analyses are very useful in the identification of clinical isolates of M. canis and M. gypseum.

Monascus fungi are commonly used for a variety of food products in Asia, and are also known to produce some biologically active compounds. Since the use of Monascus is expected to increase in food industries, strain-level identification and management of Monascus will be needed in the near future. In the present study, random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis was applied for this purpose. Evaluations of the analysis stability revealed that reproducible results could be obtained, although template DNA fragmentation could influence the resulting RAPD pattern. RAPD analysis using 15 Monascus strains consisting of four species, M. ruber, M. pilosus, M. purpureus, and M. kaoliang showed that each strain generated a unique RAPD pattern, which allows strain-level identification of Monascus. In addition, the phylogenetic tree constructed from RAPD patterns reflected M. ruber-M. pilosus and M. purpureus-M. kaoliang clusters inferred from both ITS and beta-tubulin gene sequences, which indicated that the RAPD pattern could reflect their phylogenetic traits to a certain extent. On the other hand, RAPD analysis did not support the monophyletic clustering of the four Monascus species used in this study, which suggests the necessity of reexamination of species boundaries in Monascus.

Objective To investigate intraspecific and interspecific variation within Malassezia iso-lates from patients with pityriasis versicolor by random amplified polymorphic DNA (RAPD) analysis, to learn the difference between RAPD analysis and physiological and biochemical methods in the typing of Malassezia species, and to explore the relationship between RAPD patterns and Malassezia species. Methods A total of 47 Malassezia isolates were obtained from 34 patients with pityriasis versicolor, and they were classified into 5 species by morphological, physiological and biochemical features, I.e., M. Fin'fur, M. Obtusa, M. Globosa, M. Restricta and M. Sympodialis. Genomic DNA was extracted from the 47 clinical isolates and 10 reference strains (including 7 species) of Malassezia. PCR was performed using 4 random primers including S22, S24, S25 and S33. RAPD patterns were analyzed by NTSYS software and dendrogram was autogenerated. Results Genomic DNA of most strains was successfully amplified with four primers, espe-cially with primers S22 and S24 that resulted in rather stable and clear DNA bands. A total of 82 fragments were amplified from all tested strains. These strains showed both interspecifie and intraspecific variation. Multiple swains were isolated from different body sites of 4 patients and identified into different species by biochemical and morphological typing; those swains from same hosts occupied contiguous positions in the dendrogram and exhibited a high genetic convergence. Conclusion The phenomenon that different strains from a co-host show a high genetic convergence indicates that species specificity and evolution of Malassezia are closely related to its hosts. Key words: Tinea versicolor; Malassezia; Random amplified polymorphic DNA technique

Genotyping of Ganoderma species by improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis

The randomly amplified polymorphic DNA (RAPD) assay was used to identify genetic polymorphisms in three clonal populations of the freshwater bryozoan, Cristatella mucedo, a species with few useful biochemical genetic markers. Of the 19 decamer oligonucleotide primers screened, 13 gave clear, reproducible RAPD profiles. Clonal population structure was evident, and one clone was dominant at each site. Cluster analysis grouped populations from more distant localities separately (Thames Valley and Norfolk), whereas populations from the Thames Valley clustered together. However, even at the regional scale a high degree of relatedness pertained. This work is one of the first RAPD studies of natural populations, and demonstrates the suitability of the technique for examining population structure and geographic variation in clonal taxa.

A Simplified Analysis of Different Escherichia coli Strains by Using RAPD Technique

Failure of bacteriophage typing to detect an inter-hospital outbreak of methicillin-resistant Staphylococcus aureus (MRSA) in Zagreb subsequently identified by random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE).

Molecular characterization of 18 species belonging to the genus Microsporum isolated from human and animal dermatophytosis was carried out by random amplification of polymorphic DNA (RAPD) and Southern hybridization analyses. All of these 18 Microsporum species showed different band patterns specific to each species in RAPD analysis. Southern hybridization analysis using C3 probe derived from a RAPD band from Arthroderma otae, teleomorph of M. canis, revealed distinct bands in 14 of the 18 Microsporum species but not in the other four species. The sizes of the hybridized bands of the 14 species were specific to each species, except three species (A. otae, M. equinum and M. ferrugineum) showing bands of the same size. These findings indicate that the RAPD and the Southern hybridization analyses could be effective to differentiate the species of Microsporum.

Differentiation of avian pathogenic Escherichia coli (APEC) strains by random amplified polymorphic DNA (RAPD) analysis

The usefulness of random amplification of polymorphic DNA (RAPD) analysis for typing Indian strains of M. tuberculosis was investigated. M. tuberculosis H37Rv, M. tuberculosis DT and 42 clinical isolates of M. tuberculosis were subjected to RAPD-PCR using 7 random decamer primers. All 7 primers were found to be differentiated and produced specific RAPD profiles. The polymorphic amplicons served as RAPD markers for M. tuberculosis. The dendrograms, obtained by different primers, showed the discriminatory ability of the primers. RAPD analysis provided a rapid and easy means of identifying polymorphism in M. tuberculosis isolates, and it was found to be a valuable alternative epidemiological tool. In addition, the results of the present study showed heterogeneity in the M. tuberculosis strains in the population studied.

Pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis were used to compare 21 Mycoplasma gallisepticum strains and five M. imitans strains. Each strain of M. gallisepticum typed by PFGE and RAPD methods was genetically quite unique and RAPD and PFGE fingerprinting enabled strain characterization. Relationships between the M. gallisepticum and M. imitans strains were established and dendrograms were drawn from PFGE and RAPD patterns. PFGE group A and RAPD group D were significantly associated with M. imitans strains (P < 0.05). Three M. imitans strains shared the same PFGE and RAPD patterns. The two M. gallisepticum vaccine strains had singular PFGE and RAPD patterns. Thus, PFGE and RAPD can be used to investigate disease outbreaks in vaccinated flocks or for epidemiological tracking. For M. gallisepticum, the RAPD and PFGE discriminatory powers were superior to 0.95 and the in vitro, in ovo and in vivo reproducibility of RAPD and PFGE was 100%. The RAPD drawback was the inconsistent band intensity complicating the interpretation of patterns, while the PFGE limit was its low typeability (86%). Thus, these two molecular typing methods seemed complementary for M. gallisepticum epidemiological studies.

Pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis were used to compare 21 Mycoplasma gallisepticum strains and five M. imitans strains. Each strain of M. gallisepticum typed by PFGE and RAPD methods was genetically quite unique and RAPD and PFGE fingerprinting enabled strain characterization. Relationships between the M. gallisepticum and M. imitans strains were established and dendrograms were drawn from PFGE and RAPD patterns. PFGE group A and RAPD group D were significantly associated with M. imitans strains (P < 0.05). Three M. imitans strains shared the same PFGE and RAPD patterns. The two M. gallisepticum vaccine strains had singular PFGE and RAPD patterns. Thus, PFGE and RAPD can be used to investigate disease outbreaks in vaccinated flocks or for epidemiological tracking. For M. gallisepticum, the RAPD and PFGE discriminatory powers were superior to 0.95 and the in vitro, in ovo and in vivo reproducibility of RAPD and PFGE was 100%. The RAPD drawback was the inconsistent band intensity complicating the interpretation of patterns, while the PFGE limit was its low typeability (86%). Thus, these two molecular typing methods seemed complementary for M. gallisepticum epidemiological studies.

Random amplified polymorphic DNA (RAPD) analysis was assessed as a method to identify polymorphic markers in dogs for potential use in linkage analysis of traits segregating in specific pedigrees. Three sets of primers were evaluated: 294 ten‐nucleotide long arbitrary primers from sets commonly used in RAPD analysis; 40 primers containing simple repeat elements; and 10 long random primers (19‐24 nucleotides). Of the 294 10‐mer RAPD primers, 220 (75%) amplified DNA segments from template DNA yielding primarily of 4‐5 DNA bands. Furthermore, only a few of the primers containing simple repeat elements produced discrete DNA fragments on amplification. On the other hand, all ten of the longer primers yielded amplification products with an average of 10 bands. Polymorphic bands were identified among the test DNA samples in 30% of reactions using 10‐mer RAPD primers, and in 50% of cases utilizing the long primers. RAPD primers with higher GC content amplified more polymorphic DNA fragments. Characteristic differences in amplification patterns were noted among breeds with several of these primers. We conclude from this study that by selecting primers with high GC content RAPD analysis can be successfully applied to identify polymorphic markers even in relatively inbred dog populations, and offers a promising method to search for markers linked to genetic traits of interest.

The phylogenic relationship within four species of Polygonum (including Polygonum hyrcanicum Rech. f. (three samples), Polygonum persicaria Boiss & Bushe Boiss, Polygonum avicular L., and Polygonum hydropiper L. subsp. Hydropiper ) was investigated by randomly amplified polymorphic DNA analysis (RAPD) and inter simple sequence repeat (ISSR) markers. All the species were clearly identified using 20 RAPD primers (10-mer ZO1- ZO20) and 15 ISSR primers (S1-S15). The total number of amplification products produced with 20 primers of both markers was 315. Genetic distance was calculated in order to construct phylogenic dendrogram or phylogram of closely related samples. Results indicate that the genetic distance between two samples of P. hyrcanicum originated from Sari and Goharbaran was considered to be short (0.622) and their RAPD and ISSR banding patterns were quite similar to each other also there is a close relationship between these two samples of Polygonum with P. avicular (0.848). The species P. hydropiper is not only far from two samples of P. hyrcanicum but also, has different patterns of RAPD and especially ISSR profiles compared to other samples. It is also interesting that P. hyrcanicum , gathered from Veresk, has no close relationship with other pairs of P. hyrcanicum . More chemotaxonomic investigation is needed to support the phylogenetic classification trees. Key words : Polygonum, Polygonaceae, molecular markers, P. hyrcanicum, P. persicaria, P. avicular, P.hydropiper

Genotyping of Mycoplasma gallisepticum and M. synoviae by Amplified Fragment Length Polymorphism (AFLP) analysis and digitalized Random Amplified Polymorphic DNA (RAPD) analysis