Genetic diversity of Iranian clinical isolates of Mycobacterium tuberculosis

In the current study we aimed to execute a rather less complicated molecular tying method, i.e. the random amplification of polymorphic DNA (RAPD) analysis to find the heterogeneity of Iranian strains of Mycobacterium tuberculosis. The isolates comprised a total of 96 strains of M. tuberculosis collected from clinical specimens of patients in Isfahan and Tehran. The isolates were assigned to the species M. tuberculosis by the key conventional and molecular methods. They were then subjected to RAPD analysis by four arbitrary primers, namely, the primers 27F, 1525R, MS- GF and INS-2. They were then evaluated for the number and intensity of the band patterns. The RAPD profiles of the Iranian isolates showed a degree of heterogeneity which varied based on the primer used. However, analysis of the isolates by primer INS-2 revealed the highest degree of diversity yielding 31 distinguishable RAPD types. RAPD analysis provides a rapid and easy means of identifying heterogeneity among the M. tuberculosis isolates. This typing system might be considered a valuable alternative molecular typing for countries with limited resources provided that the reproducibility and reliability of the method is carefully assured.     

New restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus

In this study, we isolated and tested restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus based on PCR products amplified by the random amplified polymorphic DNA (RAPD) primer R108. Four DNA fragments, Afd, Af5, Af4, and Af4A, were amplified. Fragments Afd and Af5 were 85% and 88% identical at the DNA level to part of the Afut1 retrotransposon from A. fumigatus. Fragment Af4A is a duplication of fragment Af4 and both showed similarity at the amino acid level with endonucleases from other fungal retrotransposons. We used both RAPD with primer R108 and RFLP assays with Afut1, Afd, and Af4A, to determine the genetic relatedness of clinical isolates of A. fumigatus isolated sequentially from four patients colonized with A. fumigatus. The combination of these different methods suggested that the isolates infecting the four patients were not identical.     

Genetic characterization of Brazilian strains of Aspergillus flavus using DNA markers

The Aspergillus genus belongs to a filamentous fungal group characterized by wide dispersion in the environment. Some species are associated with diseases, especially in immunocompromised patients, while others are of economical importance due to aflatoxin production or biotechnological applications. Its species identification is nowadays performed by traditional techniques combined with molecular markers, resulting in a higher efficiency of isolate characterization. In the present study, internal transcribed spacer, inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) molecular markers were used, with the aim of genetically characterizing strains of Aspergillus flavus and strains of other species of the A. flavus group. High genetic diversity was revealed by RAPD and by ISSR, in which the use of the (GACA)4 primer yielded a higher diversity than with the (GTG)5 primer, although the latter showed a characteristic banding profile for each species. These data were used to create a similarity matrix for the construction of dendrograms by means of the UPGMA method. The ISSR and RAPD profiles showed that among the strains previously identificated as A. flavus, one should be A. oryzae, one A. parasiticus and two A. tamarii. On the other hand, a strain previously identified as A. parasiticus should be A. flavus. All these strains were retested by traditional methods and their new species identification was confirmed. These results strongly support the need for using molecular markers as an auxiliary tool in differentiating fungal species and strains.     

Use of multiple primers in RAPD analysis of clonal organisms provides limited improvement in discrimination.

Randomly amplified polymorphic DNA (RAPD) analysis using two or more primers has been reported to provide additional discriminatory ability over one primer used individually. This may be of particular application in epidemiological typing of clonal organisms, such as Shiga toxin-producing E. coli O157, where strain differentiation can be difficult. Using four arbitrary primers individually, and in all possible permutations, E. coli O157 isolates and other arbitrarily chosen E. coli strains were typed using RAPD analysis. For most nonclonal strains, the use of two primers resulted in increased differentiation between isolates; however, more than two primers did not increase further the discriminatory capacity. E. coli O157 isolates that produced virtually identical profiles using one primer did not show increased differentiation when using two or more primers, demonstrating that in some cases, where strains of an organism are highly related, there is limited advantage to using more than one primer in RAPD analysis.     

RAPD typing for distinguishing species and strains in the genus Listeria

The randomly amplified polymorphic DNA (RAPD) technique was employed in the development of a typing protocol for Listeria isolates, particularly Listeria monocytogenes strains. A single strain of L. monocytogenes was used and 200 random decamer primers were screened for their discriminatory abilities by visualizing the amplification products electrophoretically. Three candidate primers displaying potentially useful banding patterns were selected and tested against 52 L. monocytogenes strains, encompassing 11 serotypes, and 12 other strains representing five other Listeria spp. Thirty-four banding profiles were obtained with one particular primer. RAPD analysis allowed differentiation between Listeria spp. and was found to further subdivide strains of the same serotype. Where only one primer was used strains from different serotypes were occasionally found to produce identical banding profiles. RAPD analysis, which in our hands proved to be reproducible, shows much promise as a molecular alternative to traditional L. monocytogenes typing protocols.     

Genetic diversity analysis of isolates belonging to Bordetella genus

In this study, RandomAmplified Polymorphic DNA (RAPD) method was used to track differences among human and animals isolates of eight known species of Bordetella genus. One hundred representative strains of these species from international and Polish bacterial collections were genotyped according to RAPD protocols using primer 1254 or 1247. Problems with reproducibility and discriminatory power, frequently discussed in literature, have been overcome by precise optimization procedure, which allowed to achieve reliable conditions for Bordetella species analysed. This study proved high potential of RAPD method using primers 1247/1254 for intra-species differentiation of B. bronchiseptica and B. hinzii isolates. Additionally RAPD method has been found to be useful for confirming B. avium and B. holmesii identification.     

Differentiation of <Emphasis Type="Italic">Aspergillus niger</Emphasis> by random amplification of polymorphic DNA

The randomly amplified polymorphic DNA (RAPD) patterns of whole-cell lysates from five Aspergillus niger isolates, including one reference strain, two isolated from deep freeze, and two environmental strains from soil and plant infections, were investigated. PCR-RAPD analysis of genomic DNA was performed using eight primers (Tube-A1, Tube-A6, Tube-A17, Tube-B8, Tube-B11, Tube-B15, Tube-C5, Tube-C6). The RAPD assay discriminated between all strains. Comparison of deep freeze isolates showed identical RAPD patterns in some of the reference and environmental isolates. The data indicates that the RAPD technique is useful for fingerprinting A. niger.     

RAPD discrimination of Agaricus bisporus mushroom cultivars

Cultivars of the white button mushroom Agaricus bisporus are difficult to differentiate, which has made strain protection problematic for this crop species. We have used RAPDs to discriminate between 26 strains of A. bisporus, 24 of which were commercial cultivars, and to characterise the genetic relatedness of these strains. Using 20 primers, 211 RAPD markers were identified and used in hierarchical cluster, patristic distance and parsimony analyses. All strains could be differentiated using the aggregated primer data. Although no one primer could differentiate all 26 strains, several individual primers yielded unique fingerprints for a variety of strains. The greatest differences (up to 28% variation) were observed in comparisons with or between two wild collections of A. bisporus. Quondam cultivars, commercial brown and off-white varieties proved more variable than the widely grown 'hybrid' types. Of the 15 hybrid varieties analysed, only one differed substantially (20% or more variable). The patristic and parsimony analyses both demonstrated the gross similarity of the hybrids, many of which appear to be essentially derived varieties from two original hybrid cultivars. RAPD analyses can assist mushroom strain identification and could play a role in the protection of novel cultivars.     

<Emphasis Type="Italic">Trichophyton rubrum</Emphasis> and <Emphasis Type="Italic">Trichophyton interdigitale</Emphasis>: Genetic Diversity Among Species and Strains by Random Amplified Polymorphic DNA Method

Onychomycosis is a common condition that represents up to 50% of all nail problems and 30% of all cases of dermatophytoses. Trichophyton rubrum and Trichophyton interdigitale are the most common agents involved in this condition. In cases of recurrent post-treatment onychomycosis, strain fingerprinting could reveal whether the original isolate is responsible, a new strain has been acquired or if multiple strains are involved. The aim of this study was to evaluate the efficacy of the RAPD method for species and strain differentiation of T. rubrum and T. interdigitale obtained from patients with subungeal distal-lateral onychomycosis. A set of 86 strains of onychomycosis causative dermatophytes were submitted to species differentiation and strain typing by RAPD method with two previously described primers. Both primers proved capable of strain differentiation when tested for each species. Nineteen molecular profiles were configured for T. rubrum isolates with primers 1 and 6. For T. mentagrophytes, ten molecular profiles were configured with primer 1 and twenty-one with primer 6. We found that T. interdigitale and T. rubrum species were grouped in different clusters when both primers were analyzed together. This study shows that these primers are valuable tools for strain differentiation with T. rubrum and T. intedigitale.     

Application of Random Amplified Polymorphic DNA analysis to differentiate strains of Salmonella typhi and other Salmonella species

A Random Amplified Polymorphic DNA (RAPD) fingerprinting method was developed to differentiate isolates of Salmonella serotype typhi ( S. typhi ) and other Salmonella isolates. A panel of five primers was used to examine 63 isolates of Salm. typhi , including 56 strains isolated in Taiwan and seven strains obtained abroad. Twenty-one RAPD types were revealed using the RAPD fingerprinting method. An RAPD with primer 6032 yielded a polymorphism in a 350 bp fragment that differentiated the attenuated vaccine strain Salm. typhi Ty21a from the rest of the Salm. typhi strains. Strains of Salm. typhi were divided into five types with primer D14307. Primer D14307 also proved capable of discrimination among 65 other Salmonella isolates representing 42 different serotypes. The bacterial DNA used in this RAPD protocol was obtained using a commercially available DNA extraction kit (GeneReleaser). The DNA of various strains of Salmonella from this simple extraction procedure could be discriminated within a few hours using the RAPD technique.     

Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques

The possibility of using PCR for rapid identification of food-borne Staphylococcus aureus isolates was evaluated as an alternative to the API-Staph system. A total of 158 strains, 15 S. aureus, 12 other staphylococcal species, and 131 isolates recovered from 164 food samples were studied. They were phenotypically characterized by API-Staph profiles and tested for PCR amplification with specific primers directed to thermonuclease (nuc) and enterotoxin (sea to see) genes. Disagreement between the PCR results and API-Staph identification was further assessed by the analysis of randomly amplified polymorphic DNA (RAPD) profiles obtained with three universal primers (M13, T3, and T7) and 16S rDNA sequencing. Forty out of 131 isolates (31%) tested positive for PCR enterotoxin. Of these, 14 (11%) were positive for sea, 22 (17%) for sec, one (0.8%) for sed, and three (2.2%) for sea and sec. No amplification corresponding to seb nor see was obtained. Cluster analysis based on RAPD profiles revealed that most of the sec positive food isolates grouped together in three clusters. Cluster analysis combining the three RAPD fingerprints (M 13, T3, and T7), PCR-enterotoxin genotype and API-Staph profiles, grouped the nuc PCR positive isolates together with S. aureus reference strains and the nuc PCR negative isolates with reference strains of other staphylococcal species. The only nuc PCR positive food isolate that remained unclustered was a sed positive strain identified by 16S rDNA sequence as S. simulans. The high concordance between S. aureus and nuc PCR positive strains (99%) corroborates the specificity of the primers used and the suitability of nuc PCR for rapid identification of S. aureus in routine food analysis.     

Serologischer Nachweis des Gurkenmosaik- Virus unter Verwendung vereinfachter Präzipitationsteste (Kurze Mitteilung)

A?atoxins are carcinogenic metabolites produced by Aspergillus flavus. Rice seeds may be contaminated by it at the time of harvesting or during storage. Detection of aflatoxigenic strains by TLC and analysis of genetic variability among 50 A. flavus cultures from seeds of various rice genotypes by PCR using 18 RAPD primers is reported here. About 58% isolates were aflatoxigenic whereas 42% were non-toxic. There were 246 bands and 48 haplotypes. Approximately 99% bands were polymorphic, and thus high degree of genetic variability was observed. All the primers were informative with PIC values of range 0.75–0.95. Seventeen RAPD markers were not found to be useful for the detection of aflatoxigenic A. flavus. However, one RAPD primer 3B could differentiate aflatoxigenic and non-toxigenic isolates to some extent. One allele of Primer 3B at approximately 480?bp was present in 85% aflatoxin non-producers and in 6% aflatoxigenic isolates. This information may be utilised for designing markers for differentiating toxic/non-toxic isolates of A. flavus.     

On the specificity of PCR detection of Listeria monocytogenes in food: a comparison of published primers

A total of nine pairs of primers, seven previously published and two newly developed, have been assayed for PCR detection of Listeria monocytogenes in food. They have been tested for specificity on a total of 72 strains including reference and food isolates belonging to L. monocytogenes and other species in the genus. First of all, a polyphasic approach has been carried out in order to establish a reference strain collection. They were biochemically and genetically characterized by API-Lis and randomly amplified polymorphic DNA PCR (RAPD-PCR), respectively. Random amplification of DNA was performed with M13, T7 and T3 universal primers and a data bank was created to compile the RAPD patterns of all the analyzed strains. The UPGMA cluster analysis of RAPD profiles with primer M13 showed eight clusters at 72.3% similarity. Clusters 2 and 7 corresponded to L. monocytogenes. Clusters 1 and 6 grouped L. ivanovii strains. Clusters 3, 4, 5 and 8 corresponded to L. grayi, L. innocua, L. welshimeri and L. seeligeri, respectively. Pattern analysis revealed the existence of miss-identified reference strains which was confirmed by 16S rDNA sequence analysis. RAPD-PCR is a rapid genetic test which helped to confirm strain identity. On the basis of PCR specificity results, primers LM1-LM2 were the best combination for the detection of L. monocytogenes since they only amplified the specific fragment in strains that had been genetically and biochemically assessed as belonging to the species. Specificity of other assayed primers is discussed.     

利用RAPD技术对木耳属菌株进行分类鉴定的研究

利用ＲＡＰＤ技术对木耳属不同种和种内不同菌株进行了分子鉴定。结果表明,在供试的２６个随机引物中,有１０个引物可扩增出清晰、稳定的ＤＮＡ带型,其中引物Ｓ４和Ｓ６与供试木耳属菌株的基因组ＤＮＡ结合位点少而保守,扩增图谱具有种的特异性,可用于种的快速准确鉴定;其综随机引物的扩增ＤＮＡ多态性较强,可用于种内不同菌株的鉴定;聚类分析表明,在７５％相似水平下,可将供试的木耳属菌株分成四大类,其中有三大类各代表一个形态鉴定的种。研究结果表明了ＲＡＰＤ技术可有效地用于木耳属种或菌株的快速准确鉴定。     

利用RAPD技术对木耳属菌株进行分类鉴定的研究*

利用ＲＡＰＤ技术对木耳属不同种和种内不同菌株进行了分子鉴定。结果表明,在供试的２６个随机引物中,有１０个引物可扩增出清晰、稳定的ＤＮＡ带型,其中引物Ｓ４和Ｓ６与供试木耳属菌株的基因组ＤＮＡ结合位点少而保守,扩增图谱具有种的特异性,可用于种的快速准确鉴定;其综随机引物的扩增ＤＮＡ多态性较强,可用于种内不同菌株的鉴定;聚类分析表明,在７５％相似水平下,可将供试的木耳属菌株分成四大类,其中有三大类各代表一个形态鉴定的种。研究结果表明了ＲＡＰＤ技术可有效地用于木耳属种或菌株的快速准确鉴定。     

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.     

Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures

Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

Development of specific PCR primers for the detection of Rhizoctonia solani AG 2-2 LP from the leaf sheaths exhibiting large-patch symptom on zoysia grass

Detection of Rhizoctonia solani AG 2-2 LP isolates causing large-patch disease on zoysia grass was done using polymerase chain reaction (PCR). Specific primers were designed based on an amplified region using random amplified polymorphic DNA (RAPD)-PCR. Fifteen primers and three cultural types of R. solani AG 2-2 (types IIIB, IV and LP) were used for RAPD-PCR. The banding patterns by RAPD-PCR showed that the three cultural types were clearly distinguishable. A dendrogram constructed from the results of RAPD-PCR showed that the three cultural types of AG 2-2 clustered separately. The sequence of one PCR-amplified region which appeared only in LP isolates using primer A09 was selected for designing specific primers. Primer pair A091-F/R gave a single product from pure fungal DNA of LP isolates but not from those of the other two types (IIIB and IV), R. solani AG 1, 2-1, 2-3, 2-tulip, 3-10 and BI isolates and other turfgrass fungal pathogens. Primer pair A091-F/R also gave a single product from diseased leaf sheaths and this product was in accordance with those of pure fungal DNA of LP isolates. Primer pair A091-F/R did not yield PCR product from healthy leaf sheaths. The frequencies of detection of LP isolates from leaf sheaths of zoysia grass using PCR with primer pair A091-F/R were higher than those of the conventional isolation technique. These results showed that the PCR-based technique using specific primers A091-F/R is useful for the rapid detection of LP isolates from leaf sheaths of zoysia grass exhibiting large-patch symptoms.     

Development of a rapid identification method for Aeromonas species by multiplex-PCR

Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.