DNA Fingerprinting Assays Research Articles

Authentication of putative competitive bacterial endophytes of rice by re-isolation and DNA fingerprintingassay.

The plant-growth-promoting putative competitive endophytes offer significant benefits to sustainable agriculture. The unworthy opportunistic and passenger endophytes are inevitable during the isolation of putative competitive endophytes. This study aimed to discriminate the putative competitive endophytes undoubtedly from the opportunistic and passenger endophytes. The newly isolated endophytes from field-grown rice were inoculated to 5-days old rice seedlings under gnotobiotic conditions. Re-isolation of the inoculated strains from the root surface, inner tissues of the whole plant, root and shoot was performed after 5-days. All the re-isolated colonies were compared with native isolates for homology by BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) DNA fingerprints. The results revealed that the putative competitive endophytes (RE25 and RE10) showed positive for re-isolation and BOX and ERIC fingerprints for the whole plant, root and shoot. The opportunistic (RE27 and RE8) and passenger endophytes (RE44 and RE18) failed in re-isolation either from root or shoot. The epiphytes (ZSB15 and Az204) showed negative for endophytic re-isolation and positive for surface colonization. This modified procedure can discriminate the putative competitive endophytes from others. Eliminating the opportunistic and passenger endophytes and epiphytes early by this method would help develop endophytic inoculants to enhance rice productivity.

A comparative assessment of DNA fingerprinting assays of ISSR and RAPD markers for molecular diversity of Saffron and other Crocus spp. in Iran

Genetic diversity among 25 accessions of Crocus L. (Iridaceae) representing 5 accession of cultivated C. sativus, and 20 accessions belonging to wild species namely, C. speciosus, C. cancellatus, C. haussknechtii, C. michelsonii, and two unknown accessions, obtained from different geographical locations of Iran were studied using 7 ISSR and 10 RAPD primers. Out of 78 bands generated from 7 ISSR primers, 71 were polymorphic (90.2%), whereas out of 94 bands generated from 10 RAPD primers 83 were polymorphic (88.3%). The average PIC value obtained with ISSR and RAPD markers was 0.366 and 0.324, respectively. Marker index (ISSR = 3.71; RAPD = 2.68) and resolving power (ISSR = 5.31; RAPD = 4.12) indicated that the ISSR markers were relatively more efficient than RAPD assay in respect of revealing the genetic diversity of Crocus species. Based on percentage of polymorphic bands; resolving power; polymorphic information content; and Marker Index, the A1 (ISSR) and OPB-12 (RAPD) primers were the most informative ones. Clusters obtained from ISSR, RAPD and a combination of both data sets did match the actual taxonomic classification. The mantel test between two Jaccard’s similarity matrices gave r = 0.862, showing very good fit correlation in between ISSR and RAPD based similarities. The analysis of molecular variance showed that the total polymorphism was largely because of within-group variance (ISSRs 57%; RAPDs 64%; Combined RAPDs + ISSRs 60%). Principal coordinates analysis of the pooled data of both the markers, also supports their UPGMA dendrogram. The results will help in future genetic improvement programme of Crocus species.

Development of multilocus sequence typing (MLST) assay for Mycoplasma iowae

Mycoplasma iowae (MI) infection is an economically and commercially important disease of turkeys. There are no sequence typing assays available for MI strain identification, the only available molecular tools for this purpose, are DNA fingerprinting assays. In addition to their low reproducibility, fingerprinting assays require isolation of the microorganism in pure culture, which is difficult for avian mycoplasma. Therefore, we propose a multilocus sequence typing (MLST) assay as the first genotyping assay for identification of MI. Based on the two available MI genomes on GenBank, 26 loci of housekeeping genes were identified and studied in a diverse sample set. Finally, six genes were selected for the newly developed MLST assay. The final sequence analysis of the six loci (total of 5019bp) (dppC, ulaA, valS, rpoC, leuS, kdpA) allowed the differentiation of 47 MI samples into 23 unique sequence types. Moreover, when only 4 loci were used to type the same set of samples, they resulted in 20 unique sequence types. Analysis of phylogenetic trees and clonal groups generated by MLST displayed a high degree of agreement with geographical and temporal information of the tested samples. MLST is a highly reproducible molecular epidemiology assay that can be used to identify positive clinical cases directly from DNA samples. Therefore, it provides a useful tool allowing for better identification, control and eradication efforts.

Deciphering the factors associated with the colonization of rice plants by cyanobacteria.

Cyanobacteria-rice plant interactions were analyzed using a hydroponics experiment. The activity of plant defense and pathogenesis-related enzymes, scanning electron microscopy, growth, nitrogen fixation (measured as ARA), and DNA fingerprinting assays proved useful in illustrating the nature of associations of cyanobacteria with rice plants. Microscopic analyses revealed the presence of short filaments and coiled masses of filaments of cyanobacteria near the epidermis and cortex of roots and shoot tissues. Among the six cyanobacterial strains employed, Calothrix sp. (RPC1), Anabaena laxa (RPAN8), and Anabaena azollae (C16) were the best performing strains, in terms of colonization in roots and stem. These strains also enhanced nitrogen fixation and stimulated the activity of plant defense/cell wall-degrading enzymes. A significantly high correlation was also recorded between the elicited plant enzymes, growth, and ARA. DNA fingerprinting using highly iterated palindromic sequences (HIP-TG) further helped in proving the establishment of inoculated organisms in the roots/shoots of rice plants. This study illustrated that the colonization of cyanobacteria in the plant tissues is facilitated by increased elicitation of plant enzymes, leading to improved plant growth, nutrient mobilization, and enhanced plant fitness. Such strains can be promising candidates for developing "cyanobacteria colonized-nitrogen-fixing rice plants" in the future.

Bovine salmonellosis in Northeast of Iran: Frequency, genetic fingerprinting and antimicrobial resistance patterns of Salmonella spp.

ObjectiveTo evaluate serovar and antimicrobial resistance patterns of Salmonella spp isolated from healthy, diseased and necropsied cows and calves in this observational study. MethodsNineteen isolates recovered from feces and tissues of salmonellosis-affected animals of two commercial farms in north-east of Iran. In second part of the study, the two farms were sampled 4 times with an interval of 2 month. The samples included calves' feces, adult cows' feces, feeds, water, milk filters, and milk fed to calves. Five Salmonella were isolated from 332 fecal samples collected from calves and peri-parturient cows. No Salmonella was recovered from water, feed, milk filers and milk fed to calves. ResultsSalmonella Typhimurium was the most frequently isolate among all sero-groups. S. Dublin was only accounted for 8% (two out of 24) of isolates. Isolated Salmonella strains were used for the ERIC PCR DNA fingerprinting assay. Our results grouped Salmonella isolates into 3 clusters, suggesting that specific genotypes were responsible for each sero-group of Salmonella. The results also revealed diversity among Salmonella isolates in cluster III (sero-group B). Eighteen out of 19 Salmonella spp. were resistant to oxytetracycline. Five isolates out of 19 showed more than one drug resistance. Multi-drug resistance was seen only among Salmonella Typhimurium isolates. Enrofloxacin was the most susceptible antibiotic against all isolates in this study. ConclusionThe emergence of multiple antibiotic-resistant strains of Salmonella Typhimurium should be of great concern to the public. No correlation between ERIC fingerprinting and resistance patterns of Salmonella isolates was found, which indicates resistance to antimicrobial agents was not related to specific genetic background.

Value of quantitative DNA fingerprinting in classification of second breast tumors and association with outcome.

10552 Background: Management of second breast tumors is challenging. Recent data suggest that second breast tumors may be associated with worse outcomes compared to initial tumors, implying that some second tumors may be recurrences or metastases (mets). We hypothesize that quantitative DNA fingerprinting may improve classification of second breast tumors as independent primaries vs mets, affecting prognosis and treatment. Methods: We modified our published DNA fingerprinting assay into a high throughput platform to generate scores quantifying genetic differences between breast tumors. Higher scores reflect difference. With IRB approval, we collected samples and data from 34 patients (pts): 20 had ipsilateral lymph node (LN) mets and 19 had > 1 breast tumor. We modeled independent primaries using pairs of tumors from different pts. We modeled mets using pairs of tumors and LN mets from the same pt. We generated a receiver operator curve (ROC) of the scores to classify any tumor pair as independent primaries, mets, or indeterminate. We then classified tumor pairs from pts with > 1 breast tumor and followed them until disease progression. Results: We performed quantitative DNA fingerprinting on 181 tumor and LN mets samples. Scores of pairs of tumors from different pts (n = 420) averaged 0.606. Scores of pairs of tumors and their LN mets (n = 20) averaged -0.091. These scores differed (p < 10-25, student's T-test). Based on the ROC (area under the curve 0.975), scores > 0.227 were independent primaries and scores < 0.187 were mets with 95% sensitivity. For pts with > 1 breast tumor, clinical diagnosis of the second tumor (as independent primary or met) was often indeterminate (11/19 cases, 58%) and did not predict outcome. Genetic scoring decreased indeterminate cases (2/19 cases, 11%). Kaplan Meier analysis at 60 months suggested that pts with second tumors genetically classified as an independent primaries progressed later (47 vs 25 months), although this did not reach significance (p = 0.21, log rank test). Conclusions: Quantitative DNA fingerprinting improves classification of second breast tumors into independent primaries vs mets. With further refinement, this classification may also predict progression. No significant financial relationships to disclose.

Rapid discrimination and classification of the Lactobacillus plantarum group based on a partial dnaK sequence and DNA fingerprinting techniques

The Lactobacillus plantarum group comprises five very closely related species. Some species of this group are considered to be probiotic and widely applied in the food industry. In this study, we compared the use of two different molecular markers, the 16S rRNA and dnaK gene, for discriminating phylogenetic relationships amongst L. plantarum strains using sequencing and DNA fingerprinting. The average sequence similarity for the dnaK gene (89.2%) among five type strains was significantly less than that for the 16S rRNA (99.4%). This result demonstrates that the dnaK gene sequence provided higher resolution than the 16S rRNA and suggests that the dnaK could be used as an additional phylogenetic marker for L. plantarum. Species-specific profiles of the Lactobacillus strains were obtained with RAPD and RFLP methods. Our data indicate that phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or DNA fingerprinting assays.

Isolation and characterization of microsatellite loci from blue-footed boobies (Sula nebouxii)

Blue-footed boobies (Sula nebouxii) are socially monogamous, colonial seabirds exhibiting intra-specific nest parasitism and extra-pair copulations. Prior DNA fingerprinting assays failed to detect extra-pair offspring in the nests of congeners, and the rate of intra-specific nest parasitism has not been estimated using molecular techniques. We describe the development and characterization of 11 microsatellite DNA loci, tested using 31 individuals collected on Isla Isabel, Nayarit, Mexico. The number of alleles per locus ranged from three to 22, averaging seven; total exclusionary power of the microsatellite panel was 0.99; no loci deviated from Hardy-Weinberg equilibrium; and we did not detect linkage disequilibrium following Bonferroni correction. This microsatellite panel will facilitate future studies of nest parasitism and extra-pair paternity in blue-footed boobies.

Retrotransposon-based S-SAP as a platform for the analysis of genetic variation and linkage in globe artichoke

A high copy number of retrotransposon sequences are present and widely dispersed in plant genomes. Their activity generates a considerable degree of sequence polymorphism. Here, we report the cloning of CYRE-5, a long-terminal repeat carrying retrotransposon-like sequence in Cynara cardunculus L., and its exploitation to develop a DNA fingerprinting assay across 22 accessions, including both cultivated (globe artichoke and cultivated cardoon) and wild (wild cardoon) types. The effectiveness of the sequence-specific amplified polymorphism (S-SAP) platform is compared with that of amplified fragment length polymorphism (AFLP). A genetic linkage analysis, based on a hybrid population between 2 globe artichoke varietal types, resulted in the inclusion of 29 S-SAP loci in the core genetic map, confirming their dispersed distribution across the globe artichoke genome.

The utilization of RAPD-PCR for identifying thermophilic and mesophilic Bacillus species.

A random amplified polymorphic DNA fingerprinting assay has been optimized that is able to discriminate between numerous thermophilic and mesophilic bacillus species and strains. Included in the analyses are thermophilic (able to grow at 55 degrees C) strains of Bacillus stearothermophilus, B. kaustophilus, B. coagulans, B. sphaericus, B. thermodenitrificans, B. thermocatenulatus, B. thermoleovorans, B. licheniformis, B. brevis, B. thermoglucosidasius, B. caldolyticus, B. caldotenax, B. caldovelox, B. thermocloacae and B. smithii. Mesophilic strains of B. pumilus, B. subtilis, B. megaterium, B. circulans, B. cereus and B. mycoides can also be used for fingerprinting with the assay. Increasing the concentration of primer from 0.2 to 2.0 microM is shown to have a significant effect on increasing the number of amplification products that can be used for the discrimination or identification of individual strains or species. It is suggested that this may be a general way of improving the resolution of a RAPD protocol. The optimized conditions have been used successfully to trace B. stearothermophilus, B. licheniformis and other bacillus species and strains in an industrial setting.

Identification to the species level and differentiation between strains of Aspergillus clinical isolates by automated repetitive-sequence-based PCR.

A commercially available repetitive-sequence-based PCR (rep-PCR) DNA fingerprinting assay adapted to an automated format, the DiversiLab system, enables rapid microbial identification and strain typing. We explored the performance of the DiversiLab system as a molecular typing tool for 69 Aspergillus isolates (38 A. fumigatus, 15 A. flavus, and 16 A. terreus isolates) had been previously characterized by morphological analysis. Initially, 27 Aspergillus isolates (10 A. fumigatus, 9 A. flavus, and 8 A. terreus isolates) were used as controls to create a rep-PCR-based DNA fingerprint library with the DiversiLab software. Then, 42 blinded Aspergillus isolates were typed using the system. The rep-PCR-based profile revealed 98% concordance with morphology-based identification. rep-PCR-based DNA fingerprints were reproducible and were consistent for DNA from both hyphae and conidia. DiversiLab dendrogram reports correctly identified all A. fumigatus (n = 28), A. terreus (n = 8), and A. flavus (n = 6) isolates in the 42 blinded Aspergillus isolates. rep-PCR-based identification of all isolates was 100% in agreement with the contiguous internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) sequence-based identification of the respective isolates. Additionally, the DiversiLab system could demonstrate strain-level differentiation of A. flavus and A. terreus. Automated rep-PCR may be a time-efficient, effective, easy-to-use, novel genotyping tool for identifying and determining the strain relatedness of fungi. This system may be useful for epidemiological studies, molecular typing, and surveillance of Aspergillus species.

Determination of Intronic Sequences Adjacent to an Exon Using Polymerase Chain Reaction and Genomic DNA Library Constructed by TA Cloning

To evaluate serovar and antimicrobial resistance patterns of Salmonella spp isolated from healthy, diseased and necropsied cows and calves in this observational study.Nineteen isolates recovered from feces and tissues of salmonellosis-affected animals of two commercial farms in north-east of Iran. In second part of the study, the two farms were sampled 4 times with an interval of 2 month. The samples included calves' feces, adult cows' feces, feeds, water, milk filters, and milk fed to calves. Five Salmonella were isolated from 332 fecal samples collected from calves and peri-parturient cows. No Salmonella was recovered from water, feed, milk filers and milk fed to calves.Salmonella Typhimurium was the most frequently isolate among all sero-groups. S. Dublin was only accounted for 8% (two out of 24) of isolates. Isolated Salmonella strains were used for the ERIC PCR DNA fingerprinting assay. Our results grouped Salmonella isolates into 3 clusters, suggesting that specific genotypes were responsible for each sero-group of Salmonella. The results also revealed diversity among Salmonella isolates in cluster III (sero-group B). Eighteen out of 19 Salmonella spp. were resistant to oxytetracycline. Five isolates out of 19 showed more than one drug resistance. Multi-drug resistance was seen only among Salmonella Typhimurium isolates. Enrofloxacin was the most susceptible antibiotic against all isolates in this study.The emergence of multiple antibiotic-resistant strains of Salmonella Typhimurium should be of great concern to the public. No correlation between ERIC fingerprinting and resistance patterns of Salmonella isolates was found, which indicates resistance to antimicrobial agents was not related to specific genetic background.

The utilization of RAPD-PCR for identifying thermophilic and mesophilic Bacillus species

A random amplified polymorphic DNA fingerprinting assay has been optimized that is able to discriminate between numerous thermophilic and mesophilic bacillus species and strains. Included in the analyses are thermophilic (able to grow at 55°C) strains of Bacillus stearothermophilus, B. kaustophilus, B. coagulans, B. sphaericus, B. thermodenitrificans, B. thermocatenulatus, B. thermoleovorans, B. licheniformis, B. brevis, B. thermoglucosidasius, B. caldolyticus, B. caldotenax, B. caldovelox, B. thermocloacae and B. smithii. Mesophilic strains of B. pumilus, B. subtilis, B. megaterium, B. circulans, B. cereus and B. mycoides can also be used for fingerprinting with the assay. Increasing the concentration of primer from 0.2 to 2.0 μM is shown to have a significant effect on increasing the number of amplification products that can be used for the discrimination or identification of individual strains or species. It is suggested that this may be a general way of improving the resolution of a RAPD protocol. The optimized conditions have been used successfully to trace B. stearothermophilus, B. licheniformis and other bacillus species and strains in an industrial setting.

Assessment of genetic diversity by DNA profiling and its significance in silkworm, Bombyx mori

Silkworm genetic resources that are being maintained in different countries are yet to be adequately tapped to develop elite varieties that are suited to different agro-eco-climatic conditions of countries like India. This is mostly due to unavailability of efficient protocols that could uncover usable genetic variability in silkworms. Molecular markers are known to provide unambiguous estimates of genetic variability of populations since they are independent of confounding effects of environment. The DNA fingerprinting assays, based on random amplified polymorphic DNA (RAPD) and banded krait minor satellite DNA (Bkm) 2(8) multilocus probes, which successfully characterise the diverse silkworm genotypes at their DNA level, are described. The use of these two DNA fingerprinting assays in estimation of within- and between-population genetic diversity is discussed.

Polymerase Chain Reaction-Based DNA Fingerprinting for Identification of Streptococcus and Enterococcus Species Isolated from Bovine Milk

A total of 353 isolates including 304 isolates from bovine milk belonging to 8 Streptococcus and Enterococcus species, 14 American Type Culture Collection reference strains, 10 isolates from bovine milk that had atypical biochemical profiles, and 25 bovine milk isolates from Vermont were examined by polymerase chain reaction-based DNA fingerprinting. A single short oligonucleotide primer of eight bases (5′-GTAACGCC-3′) was used to produce a characteristic spectrum of amplified DNA fragments. Amplified DNA fragments were visualized following agarose gel electrophoresis and analyzed by scanning laser densitometry. Amplified DNA fragments were categorized as either primary, secondary, or variable fragments. DNA fragments observed in all isolates within a species were designated as primary [optical density > 0.3 absorbance units (AU)] and secondary fragments (optical density > 0.15 AU), while infrequent DNA fragments were designated as variable fragments (optical density >0.15 AU). Examination of fingerprint patterns revealed that each of the species evaluated possessed a unique fingerprint profile. A simple identification scheme was developed based on the occurrence of primary and secondary DNA fragments for a species. The polymerase chain reaction-based DNA fingerprinting assay was simple to perform, reliable, and reproducible. Results of this study indicate that DNA fingerprinting using polymerase chain reaction has the potential to be developed as a routine method for species identification of bacteria.

Serological Responses of Salmonella Dublin Carrier Cows

To evaluate serovar and antimicrobial resistance patterns of Salmonella spp isolated from healthy, diseased and necropsied cows and calves in this observational study.Nineteen isolates recovered from feces and tissues of salmonellosis-affected animals of two commercial farms in north-east of Iran. In second part of the study, the two farms were sampled 4 times with an interval of 2 month. The samples included calves' feces, adult cows' feces, feeds, water, milk filters, and milk fed to calves. Five Salmonella were isolated from 332 fecal samples collected from calves and peri-parturient cows. No Salmonella was recovered from water, feed, milk filers and milk fed to calves.Salmonella Typhimurium was the most frequently isolate among all sero-groups. S. Dublin was only accounted for 8% (two out of 24) of isolates. Isolated Salmonella strains were used for the ERIC PCR DNA fingerprinting assay. Our results grouped Salmonella isolates into 3 clusters, suggesting that specific genotypes were responsible for each sero-group of Salmonella. The results also revealed diversity among Salmonella isolates in cluster III (sero-group B). Eighteen out of 19 Salmonella spp. were resistant to oxytetracycline. Five isolates out of 19 showed more than one drug resistance. Multi-drug resistance was seen only among Salmonella Typhimurium isolates. Enrofloxacin was the most susceptible antibiotic against all isolates in this study.The emergence of multiple antibiotic-resistant strains of Salmonella Typhimurium should be of great concern to the public. No correlation between ERIC fingerprinting and resistance patterns of Salmonella isolates was found, which indicates resistance to antimicrobial agents was not related to specific genetic background.