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PubMed2003

[The application of RAPD technology in genetic diversity detection of Jute].

The fingerprints of 10 species including 27 accessions in genus Corchorus were investigated with the technique of RAPD. Twenty-five primers were screened from 119 random primers, and a total of 329 DNA fragments were amplified ranging from 0.3-3.0 kb, 253 (87.78%), which were polymorphic. The average number of DNA band produced by each primer was 13.16. UPGMA cluster analysis and Nei's similarity coefficients were carried out and a dendrogram was constructed using software Biol D++. The results showed as follows: (1) There were abundant genetic diversities among 15 wild species and 12 cultivated species in Corchorus with genetic similarity coefficients ranging from 0.49-0.98. (2) The accessions could be clustered into three groups at cultivated species, and their close wild species were obviously different from wild species genetically. (3) At the level of D = 0.850, 27 accessions of Jute could be classified into ten groups, including C. sestuans, C. tridens, C. fascicularis, C. psendo-olitorius, C. psendo-capsularis, C. tilacutaris, Tian Jute (untitled), C. capsularis, C. olitorius and C. uriticifolius. Among which C. capsularis presented closer relationship with C. olitorius and further relationship with C. uriticifolius. The results matched well with that of the morphologic classification. (4) According to the molecular cluster tree, C. uritifolius, Chinese Tina Jute (untitled) and C. aestuans were at the basic level, revealing that these three species could be the primary wild species of Jute. (5) The tree also showed that C. tilacularis 21C from Africa could be a ecological subspecies of C. tilacularis, whilst niannian cai, ma cai and zhu cai collected different ecological types of C. aestuans, C. capsularis from Hainan was a close wild species of round fruit Jute cultivated species, and three species of C. olitorius collected from zhangpu, Henan and Mali were close wild species of long fruit Jute cultivated species. (6) within two cultivated species, the genetic similarity coefficients in round fruit cultivated species was higher than that of in long fruit cultivated species.

Corchorus↗

PubMed2003

Cloning and analysis of the mating-type idiomorphs from the barley pathogen Septoria passerinii.

The genus Septoria contains more than 1000 species of plant pathogenic fungi, most of which have no known sexual stage. Species of Septoria without a known sexual stage could be recent derivatives of sexual species that have lost the ability to mate. To test this hypothesis, the mating-type region of S. passerinii, a species with no known sexual stage, was cloned, sequenced, and compared to that of its close relative S. tritici (sexual stage: Mycosphaerella graminicola). Both of the S. passerinii mating-type idiomorphs were approximately 3 kb in size and contained a single reading frame interrupted by one (MAT-2) or two (MAT-1) putative introns. The putative products of MAT-1 and MAT-2 are characterized by alpha-box and high-mobility-group sequences, respectively, similar to those in the mating-type genes of M. graminicolaand other fungi. The mating-type genes of S. passerinii and M. graminicolaare evolving rapidly, approximately ten times faster than the internal transcribed spacer region of the ribosomal DNA, and are not closely related to those from Cochliobolusor other loculoascomycetes in the order Pleosporales. Therefore, the class Loculoascomycetes may be polyphyletic. Furthermore, differences between the phylogenetic trees may indicate separate evolutionary histories for the MAT-1 and MAT-2 idiomorphs. A three-primer multiplex-PCR technique was developed that allowed rapid identification of the mating types of isolates of S. passerinii. Both mating types were present in approximately equal frequencies and often on the same leaf in fields in Minnesota and North Dakota. Analyses with isozyme and random amplified polymorphic DNA markers revealed that each isolate had a unique genotype. The common occurrence of both mating types on the same leaf and the high levels of genotypic diversity indicate that S. passerinii is almost certainly not an asexual derivative of a sexual fungus. Instead, sexual reproduction probably plays an integral role in the life cycle of S. passerinii and may be much more important than previously believed in this (and possibly other) "asexual" species of Septoria.

Amino Acid Sequence↗

PubMed2001

Elucidation of Listeria monocytogenes contamination routes in cold-smoked salmon processing plants detected by DNA-based typing methods.

The contamination routes of Listeria monocytogenes in cold-smoked salmon processing plants were investigated by analyzing 3,585 samples from products (produced in 1995, 1996, 1998, and 1999) and processing environments (samples obtained in 1998 and 1999) of two Danish smokehouses. The level of product contamination in plant I varied from 31 to 85%, and no L. monocytogenes was found on raw fish (30 fish were sampled). In plant II, the levels of both raw fish and product contamination varied from 0 to 25% (16 of 185 raw fish samples and 59 of 1,000 product samples were positive for L. monocytogenes). A total of 429 strains of L. monocytogenes were subsequently compared by random amplified polymorphic DNA (RAPD) profiling, and 55 different RAPD types were found. The RAPD types detected on the products were identical to types found on the processing equipment and in the processing environment, suggesting that contamination of the final product (cold-smoked salmon) in both plants (but primarily in plant I) was due to contamination during processing rather than to contamination from raw fish. However, the possibility that raw fish was an important source of contamination of the processing equipment and environment could not be excluded. Contamination of the product occurred in specific areas (the brining and slicing areas). In plant I, the same RAPD type (RAPD type 12) was found over a 4-year period, indicating that an established in-house flora persisted and was not eliminated by routine hygienic procedures. In plant II, where the prevalence of L. monocytogenes was much lower, no RAPD type persisted over long periods of time, and several different L. monocytogenes RAPD types were isolated. This indicates that persistent strains may be avoided by rigorous cleaning and sanitation; however, due to the ubiquitous nature of the organism, sporadic contamination occurred. A subset of strains was also typed by using pulsed-field gel electrophoresis and amplified fragment length polymorphism profiling, and these methods confirmed the type division obtained by RAPD profiling.

Animals↗

PubMed1996

Detection of cytosine methylation and mapping of a gene influencing cytosine methylation in the genome of Citrus.

A new method was developed to detect DNA methylation in the Citrus genome using random amplification coupled with restriction enzyme digestion. Genomic DNA from Citrus grandis (L.) Osb., Poncirus trifoliata (L.) Raf., and their F1 hybrid was amplified using 7 individual 10-mer random primers. Prior to amplification the DNA templates were digested with 2 pairs of restriction endonucleases (HpaII-MspI and (or) Sau3AI-NdeII) with different sensitivities to cytosine methylation and after PCR amplification their amplified products were further digested with the same enzymes. Using this method, it was possible to detect 28 methylation events involving 23 amplified bands with the 7 random primers and 2 pairs of enzymes. A methylation polymorphism was found at a Sau3AI site in a 1.2-kb band amplified with one primer. One locus influencing cytosine methylation at this restriction site was identified through genetic analysis of a BC1 population between C. grandis and P. trifoliata and was mapped to linkage group IV using an already developed core map. This technique for detecting methylation and methylation polymorphisms is simple and should be applicable to any eukaryotic species and to many situations where it is desirable to determine whether a sequence is methylated.

Base Sequence↗

PubMed2000

Polyphasic identification of wild yeast strains isolated from Greek sourdoughs.

A total of forty-five wild yeast strains were isolated from five traditional Greek wheat sourdoughs. Strains were identified using the classical identification technique along with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole cell proteins (SDS-PAGE), Fourier transform-infrared spectroscopy (FT-IR) and the randomly amplified polymorphic DNA-polymerase chain reaction analysis (RAPD-PCR). The latter methods confirmed the classical identification. According to the results obtained, fourteen strains were identified as Saccharomyces cerevisiae strains, twenty-five as Pichia membranaefaciens strains and six as Yarrowia lipolytica.

Bread↗

PubMed2000

[Molecular analysis of space mutant line of kidney bean].

Objective. To identify the occurrence of gene mutant in mutant lines in the offspring of Kidney bean seeds under space flight condition. Method. Kidney bean seeds were carried onboard a recoverable satellite for 15 days in space and were planted on the ground after recovery. Five mutant lines showing variation in the form of leaf blade and their parents were analyzed with RAPD technique. Result. 50 random 10-mer primers were used in this study, among which 20 primers generated 180 polymorphic DNA bands, their size ranged from 200 bp to 2000 bp. 3 primers amplified obviously different bands in the DNA of mutant lines in comparison with that of the control. Conclusion. This is the first molecular analysis of the mutant lines of Kidney bean generated by space mutagenesis at DNA level. The result of RAPD analysis indicated that distinct variations were demonstrated in the DNA of mutant lines as compared with that of the original control.

Base Sequence↗

PubMed2000

Milkborne campylobacter infection in Hungary.

In April 1998, an annual 2-day animal farm sale was held in Hódmezóvásárhely, where 500 to 600 visitors consumed unpasteurized milk. The first signs of disease began 2 days after the end of the sale. Fifty-two people from a wide age range fell ill, primarily with inflammatory enteritis. These cases included 34 with Campylobacter positivity: 30 with Campylobacter jejuni and 4 with Campylobacter coli. Environmental samples (raw milk, udder swabs, and rectal swabs from 12 cows in the suspected herd) were tested 2 weeks after the first signs of the disease, and two rectal swabs were found to be positive for C. jejuni. Initially, the epidemic seemed to be sporadic and, accordingly, only 26 human and 2 animal Campylobacter isolates were reserved for randomly amplified polymorphic DNA analysis. This comparative analysis verified that fecally contaminated milk was the source of the outbreak. The DNA-banding patterns of 20 C. jejuni isolates (19 human and 1 animal) were identical. The antibiotic susceptibilities of the Campylobacter isolates were determined, and only six C. jejuni (human) isolates, one C. coli (human) isolate, and one C. jejuni (animal) isolate were resistant to tetracycline, both by disk diffusion and by E test (antimicrobial gradient strip for the quantitative determination of susceptibility or resistance of microorganisms). No plasmid was detected in these tetracycline-resistant isolates. The endotoxin production of Campylobacter isolates was examined via the cytopathogenic effect on the Vero cell line. This effect exhibited various degrees of positivity in 19 cases. Only two human C. jejuni isolates displayed + + + + positivity. Both isolates were from patients who had required antibiotic therapy and hospital care.

Animals↗

PubMed1999

Application of different genotyping methods for Pseudomonas aeruginosa in a setting of endemicity in an intensive care unit.

Colonization with Pseudomonas aeruginosa was studied by taking serial swab specimens from the oropharynges and anuses and tracheal and gastric aspirates from patients in an intensive care unit during a 10-month period in a setting of endemicity. Nineteen (10%) of the 192 patients included in the study were colonized on admission, while another 30 (16%) patients acquired P. aeruginosa while in the hospital. Typing of 353 isolates was performed by random amplified polymorphic DNA (RAPD) analysis, and 56 strains were selected for further typing by RAPD analysis, pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) analysis. By these methods, 42, 44, and 44 genotypes were found, respectively. Computer-aided cluster analysis indicated that similar groups of related isolates were obtained by each method. By taking admission periods into account, analysis of the typing results suggested cross-acquisition of P. aeruginosa for five patient pairs. The small number of transfers and the large number of genotypes found indicate that most P. aeruginosa strains were derived from the patients themselves. The numbers of observed typing patterns and band differences between related isolates were counted for each typing method. AFLP analysis with primers without a selective base proved to be the most discriminatory method, followed by PFGE, AFLP analysis (with one selective base), and RAPD analysis. On the basis of a comparison with established strain differentiation criteria for PFGE, the criteria for differentiation of P. aeruginosa by AFLP analysis are presented.

Bacterial Typing Techniques↗

PubMed2004

Genetic characterization of Listeria monocytogenes food isolates and pathogenic potential within serovars 1/2a and 1/2b.

A total of 39 Listeria monocytogenes strains isolated from raw milk, smoked meat, chicken carcass and reference strains, belonging to serovars 1/2a, 4a, 1/2b, 3b and 4b, were analysed by RAPD and by polymorphisms of the virulent genes inlAB and iap. Ten isolates, belonging to serovars 1/2a and 1/2b and, collected from raw milk and smoked meat, were further tested for pathogenicity by IP injection into mice. The clustering of the 39 L. monocytogenes strains in 3 groups at 0.45 similarity level, based on molecular typing, was observed. Distribution of serovars in these clusters was in agreement with the proposed three Listeria monocytogenes lineages. Within serovar 1/2b, the 50% lethal dose (LD50) ranged from 8.4 x 10(4) to 1.7 x 10(6) cfu.ml(-1). One of the serovar 1/2b strains, isolated from smoked meat, exhibited the lowest virulence potential evaluated by LD50 and by mean time to death (MTD) and, from this point of view, was completely different from the other strains. Our results suggest the existence of heterogeneity in virulence levels within serovars 1/2a and 1/2b. However, when comparing the isolates based on genotyping, virulence indicators and food origin, no relation could be assessed.

Animals↗

PubMed2001

Diversity among strains of Cryptococcus neoformans var. gattii as revealed by a sequence analysis of multiple genes and a chemotype analysis of capsular polysaccharide.

We demonstrated the diversity of Cryptococcus neoformans var. gattii strains by a sequence analysis of multiple genes: (i) the intergenic spacer (IGS) 1 and 2 regions of the rRNA gene; (ii) the internal transcribed spacer (ITS) region, including 5.8S of the rRNA gene; (iii) TOP1 (topoisomerase); and (iv) CAP59. In these studies, we compared C. neoformans var. gattii with varieties grubii, and neoformans of C. neoformans. Phylogenetic analysis indicated that both C. neoformans var. grubii and C neoformans var. neoformans are monophyletic, but C. neoformans var. gattii showed polyphyletic. C. neoformans var. gattii can be divided into three phylogenetic groups, I, II, and III, with high bootstrap support. Phylogenetic group I contains serotype B and C strains, and groups II and III include serotype B strains. Because the serotype B strains of C. neoformans var. gattii exhibited more genetic divergence, the serological characteristics and chemotypes of their capsular polysaccharide were further investigated. No remarkable difference among the serotype B strains was found in the reactivities to factor serum 5, which is specific for serotype B. The NMR spectra of the capsular polysaccharide from serotype B strains could be divided into three characteristic patterns, but the chemical shifts were very similar. These results suggested that the serotype B strain of C. neoformans var. gattii has more genetic diversity than the serotype C strain of C. neoformans var. gattii or the varieties grubii and neoformans of C. neoformans, but there was no correlation between genotype and chemotype.

Bacterial Typing Techniques↗

PubMed2005

Molecular and biochemical analysis of the galactose phenotype of dairy Streptococcus thermophilus strains reveals four different fermentation profiles.

Lactose-limited fermentations of 49 dairy Streptococcus thermophilus strains revealed four distinct fermentation profiles with respect to galactose consumption after lactose depletion. All the strains excreted galactose into the medium during growth on lactose, except for strain IMDOST40, which also displayed extremely high galactokinase (GalK) activity. Among this strain collection eight galactose-positive phenotypes sensu stricto were found and their fermentation characteristics and Leloir enzyme activities were measured. As the gal promoter seems to play an important role in the galactose phenotype, the galR-galK intergenic region was sequenced for all strains yielding eight different nucleotide sequences (NS1 to NS8). The gal promoter played an important role in the Gal-positive phenotype but did not determine it exclusively. Although GalT and GalE activities were detected for all Gal-positive strains, GalK activity could only be detected for two out of eight Gal-positive strains. This finding suggests that the other six S. thermophilus strains metabolize galactose via an alternative route. For each type of fermentation profile obtained, a representative strain was chosen and four complete Leloir gene clusters were sequenced. It turned out that Gal-positive strains contained more amino acid differences within their gal genes than Gal-negative strains. Finally, the biodiversity regarding lactose-galactose utilization among the different S. thermophilus strains used in this study was shown by RAPD-PCR. Five Gal-positive strains that contain nucleotide sequence NS2 in their galR-galK intergenic region were closely related.

Bacterial Proteins↗

PubMed1995

Use of molecular analysis in pathophysiological investigation of late-onset neonatal Escherichia coli meningitis.

We describe one case of late-onset neonatal Escherichia coli meningitis associated with urinary tract infection. Two genotypic methods revealed that the E. coli isolates obtained from blood, feces, and cerebrospinal fluid shared the same pattern, which was different from the unique pattern obtained for the isolates recovered from the urine and the tracheal aspirate. Our study supports passage of bacteria through the gastrointestinal mucosa to the bloodstream as the initial event, with subsequent meningeal localization.

Bacterial Typing Techniques↗

PubMed1999

Performance criteria of DNA fingerprinting methods for typing of Helicobacter pylori isolates: experimental results and meta-analysis.

Typing systems are used to discriminate between isolates of Helicobacter pylori for epidemiological and clinical purposes. Discriminatory power and typeability are important performance criteria of typing systems. Discriminatory power refers to the ability to differentiate among unrelated isolates; it is quantitatively expressed by the discriminatory index (DI). Typeability refers to the ability of the method to provide an unambiguous result for each isolate analyzed; it is quantitatively expressed by the percentage of typeable isolates. We evaluated the discriminatory power and the typeability of the most currently used DNA fingerprinting methods for the typing of H. pylori isolates: ribotyping, PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis, and random amplified polymorphism DNA (RAPD) analysis. Forty epidemiologically unrelated clinical isolates were selected to constitute a test population adapted to the evaluation of these performance criteria. A meta-analysis of typeability and discriminatory power was conducted retrospectively with raw data from published studies in which ribotyping, PCR-RFLP, RAPD, repetitive extragenic palindromic DNA sequence-based PCR (REP-PCR), or pulsed-field gel electrophoresis (PFGE) was used. Experimental results and the meta-analysis demonstrated the optimal typeability (100%) and the excellent discriminatory powers of PCR-based typing methods: RAPD analysis, DIs, 0.99 to 1; REP-PCR, DI, 0.99; and PCR-RFLP analysis, DIs, 0.70 to 0.97). Chromosome restriction-based typing methods (ribotyping and PFGE) are limited by a low typeability (12.5 to 75%) that strongly decreases their discriminatory powers: ribotyping, DI, 0.92; PFGE, DIs, 0.24 to 0.88. We do not recommend the use of ribotyping and PFGE for the typing of H. pylori isolates. We recommend the use of PCR-based methods.

Bacterial Typing Techniques↗

PubMed2006

Female-specific DNA sequences in ostriches.

Ostrich absence of heteromorphic sex chromosomes, unique sequences or markers located in the ostrich W-chromosome. Random amplified polymorphic DNA (RAPD) fingerprinting was carried out to investigate the sex-specific DNA sequence for sexing in ostrich. One hundred and forty random primers were used for random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). One of these primers, OPAJ-13, produced a sex-specific band only found in tested females, which was isolated and constructed into plasmids for nucleotide sequencing. A 760bp novel female-specific sequence was obtained. Two primers (OstSexOPAJ13-F and -R) were designed according to the cloned female sequence to amplify the female-specific fragment from genomic DNA of ostriches for sexing by PCR. The sex-specific band was represented in females but none were found in the males. This result showed that the sex of ostrich could be easily and effectively identified using the female-specific primers for PCR technique.

Animals↗

PubMed2002

Genomic diversity within the genus Pediococcus as revealed by randomly amplified polymorphic DNA PCR and pulsed-field gel electrophoresis.

The genomic diversity of 33 previously assigned strains from six species within the genus Pediococcus was assessed by randomly amplified polymorphic DNA (RAPD) PCR and pulsed-field-gel electrophoresis (PFGE). The RAPD PCR patterns produced by two separate random primers, termed P1 (ACGCGCCCT) and P2 (ATGTAACGCC), were compared by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm. Pattern variations between repeat samples set a strain discrimination threshold of less than 70% similarity. P1 and P2 primers alone and in combination produced 14, 21, and 28 distinct patterns, respectively. When each strain was assigned with a type strain with which it shared the highest level of similarity, both primers grouped 17 of the 27 strains to their proposed species. PFGE following genomic digestion with the restriction enzymes ApaI, NotI, and AscI produced 30, 32, and 28 distinct macrorestriction patterns, respectively. Specific DNA fragments within the NotI and AscI macrorestriction patterns for each strain were observed that allowed 27 of the 33 strains to be assigned to their proposed species. For example, following digestion with AscI, all Pediococcus parvulus strains were characterized by two DNA fragments, one of approximately 220 kb and another between 700 and 800 kb. The exceptions correlated with those observed with both RAPD PCR primers and included three P. damnosus and two P. pentosaceus strains that grew at temperatures regarded as nonpermissive for their proposed species but not for those with which they grouped.

Animals↗

PubMed2001

Biomonitoring of continuous microbial community adaptation towards more efficient phenol-degradation in a fed-batch bioreactor.

The anaerobic degradation of phenol was studied in a fed-batch culture. Nitrate was added as electron acceptor and phenol was provided three times, to a final concentration of 200 mg/l. Randomly amplified polymorphic DNA (RAPD) and terminal fraction fragment length polymorphism (T-RFLP) were used and compared in order to monitor the microbial succession in the reactor. Phenol degradation started after an initial lag phase of 14 days and was then completed within a few days. In addition, the duration of the lag phase was shortened and the degradation rate was increased after each phenol amendment. Nitrate reduction correlated with microbial growth and phenol depletion, confirming that the degradation was carried out anaerobically. Results from the DNA analysis showed that the structure of the microbial community changed after each phenol amendment. This study confirms the potential for anaerobic degradation of environmental pollutants and also confirms that microbial acclimation towards faster degradation rates occurred upon repeated substrate amendments. Furthermore, both of the DNA-based techniques described the phenol degradation-linked community shifts with similar general results. RAPD is a faster, simpler technique that gives a higher resolution and consequently reflects the shifts in the microbial community structure better, whereas T-RFLP is more suitable for phylogenetic studies.

Adaptation, Physiological↗

PubMed2000

Differentiation of Brucella species by random amplified polymorphic DNA analysis.

Random amplification of polymorphic DNA (RAPD) was used for discrimination between 46 Brucella strains and 14 representatives of the alpha-2 and alpha-1 subgroups of Proteobacteria. To evaluate a relatively quick and exact method for Brucella identification, the authors specified the most suitable conditions for RAPD amplification of Brucella DNA with two 10-mer primers, containing lower and higher percentages of G and C. The software package PHYLIP 3.1 was used for cluster analysis of the RAPD fingerprints. The optimization of RAPD conditions resulted in PCR mixes suitable for reliable typing of Brucellae. The distance-based methods (Fitch-Margoliash, UPGMA and Neighbour-joining) gave clear discrimination between Brucella species. The constructed dendrograms put Br. canis and Br. suis bv. 1 in the same cluster and differentiated Brucella strains according to their host preferences. RAPD can be useful method to distinguish related bacterial species, and under strictly established conditions the reaction appears to be a simple, quick and sensitive technique for the epidemiological investigation of brucellosis.

Brucella↗

PubMed1999

Suppression of randomly primed polymerase chain reaction products (random amplified polymorphic DNA) in heterozygous diploids.

In a study of genetic polymorphism in the gypsy moth Lymantria dispar we observed the aberrant inheritance of a random amplified polymorphic DNA (RAPD) fragment designated H11-589. This fragment was present in amplification products of F1 progeny of different crosses although it was not amplified from either parental DNA. DNA-mixing experiments revealed that the presence of DNA containing a template for another product (H11-746), amplified with the same primer, suppressed the synthesis of H11-589. The templates for both RAPD products were highly repetitive and scattered throughout the L. dispar genome. Southern hybridization and sequence analysis of H11-746 and H11-589 revealed an extensive sequence homology and an internal repetitive motif of 17 nucleotides present in both products. Interactions between templates for H11-746 and H11-589 are expected to occur during the polymerase chain reaction (PCR), offering an explanation for the suppression of the amplification of H11-589. The role of the internal repetitive motif and of the copy number of both templates in the suppression effect are discussed. Our results corroborate doubts regarding the suitability of the RAPD technique for quantitative genetic analysis, in particular where mixed populations are concerned.

Animals↗