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PubMed1996

Molecular genetic approaches to identification, epidemiology and taxonomy of non-albicans Candida species.

The reported incidence of fungal infections associated with non-albicans species from the Candida genus is increasing. Most of these infections occur in immunocompromised patients, particularly those infected with HIV. The role of molecular genetic techniques alongside the existing techniques for the identification and typing of these organisms is discussed. Species-specific genomic DNA fragments cloned from C. tropicalis and C. krusei have been developed for identification and strain typing. Analysis of tRNA profiles has been shown to be effective for the identification of C. glabrata, C. guilliermondii, C. parapsilosis and C. tropicalis. A PCR method employing primers complimentary to large ribosomal subunit genes and the lanosterol-alpha-demethylase gene has been applied for several species, including C. glabrata, C. krusei and C. tropicalis. Strain typing by comparison of genomic DNA fingerprints has been demonstrated for C. tropicalis and C. krusei following hybridisation analysis with species-specific probes. Synthetic oligonucleotide probes--which do not have to be species-specific and which can detect minor polymorphisms--have also been used for strain typing of isolates of several non-albicans species. Random amplification of polymorphic DNA (RAPD) has also been used for analysis of C. glabrata, C. lusitaniae and C. tropicalis isolates. The potential for the application of these and other techniques to Candida spp. taxonomy--and the example of a recently discovered novel species, C. dubliniensis--is discussed.

Candida↗

PubMed2002

Highly different levels of natural transformation are associated with genomic subgroups within a local population of Pseudomonas stutzeri from soil.

A highly sensitive and specific PCR-based method of monitoring 16S rRNA genes of Pseudomonas stutzeri was developed for searching P. stutzeri DNA in environmental samples. This monitoring was combined with a reliable and sensitive method for isolating P. stutzeri colony formers from soil and sediment, depending on their utilization of ethylene glycol, starch, and maltose. With these techniques, P. stutzeri populations (n = 2 to 170) were obtained from five of six sites giving positive PCR signals (including three marine sediment and two soil samples). The phylogenetic positions of isolates from the five sites, based on their 16S ribosomal DNA sequences, indicated that the environmental isolates were affiliated with different genomovars of P. stutzeri. Using the broad-host-range plasmid pNS1 with kanamycin and gentamicin resistance determinants as the transforming DNA, naturally transformable strains were identified among the isolates from all sites. For one population from soil, the genetic relationship of the 120 members was determined by randomly amplified polymorphic DNA-PCR with three PCR primers. Among the population members which are taxonomically closely related as determined by 16S sequence comparisons of group representatives, a rather high genetic diversity and a characteristic clustering into subgroups were found. Remarkably, within the population, nontransformability and different levels of transformability (a frequency between about 10(-9) and 10(-4) per cell) were often associated with distinct genetic subgroups. It is concluded that transformability is widespread among environmental P. stutzeri strains and that its specific level is a heritable trait that may vary strongly within a local population.

DNA, Ribosomal↗

PubMed2005

Identification of predominant lactic acid bacteria isolated from traditionally fermented vegetable products of the Eastern Himalayas.

Gundruk, sinki and khalpi are lactic-fermented vegetable products of Sikkim in India, and inziangsang is a fermented leafy vegetable product of Nagaland and Manipur in India. A total of 65 samples of gundruk (25), sinki (12), khalpi (25) and inziangsang (3) were analysed for microbial counts. The population of lactic acid bacteria (LAB) as well as aerobic mesophilic counts were at the level of 10(7) cfu g(-1). Yeasts were detected only in few samples of sinki and khalpi. No moulds were detected. In order to identify the predominating organisms, a total of 269 strains of LAB were isolated from gundruk, sinki, khalpi and inziangsang samples. The phenotypic characteristics of these strains were determined followed by genotyping using RAPD-PCR, repetitive element PCR and species-specific PCR techniques. The major representatives of the LAB involved in these fermentations were identified as Lactobacillus brevis, Lactobacillus plantarum, Pediococcus pentosaceus, Pediococcus acidilactici and Leuconostoc fallax.

Colony Count, Microbial↗

PubMed2005

[Study on producing area suitability of genuine traditional Chinese drugs].

The discussion centered primarily on background, basis and research ways of the study on producing area suitability of genuine traditional Chinese drugs in this paper. The authors expounded application and development of the used modem techniques. The direction and strategy of producing area suitability of genuine traditional Chinese drugs for the future were proposed and prospected aiming to provide with scientific thoughtway and guiding later production and practice.

Climate↗

PubMed2006

SCAR-based real time PCR to identify a biocontrol strain (T1) of Trichoderma atroviride and study its population dynamics in soils.

Strains of Trichoderma spp. are known for their antagonistic properties against plant pathogens, some are already on the market, others are under development. In order to launch a strain on the market its perfect identification at the species and strain levels is needed. The aim of this study is to (i) design a SCAR marker for specific identification of strain T1 of Trichoderma atroviride and (ii) monitor population dynamics of this strain in soil by real time PCR. A primer pair targeting a 141-bp fragment enabled specific detection of this strain without cross detection of autochthonous populations of Trichoderma in several field soils. In two soils, population dynamics assessed by real time PCR and the soil plate technique gave similar results. The molecular tools developed in this study satisfy the requirement for specific identification of the biocontrol strain and for detection and quantification of T. atroviride T1 population in complex environments.

DNA Fingerprinting↗

PubMed1997

Diagnosis of sibling species of Drosophila involved in the colonization of North America by D. subobscura.

To determine the effects of the recent colonization of the west coast of North America by the Palaearctic species Drosophila subobscura on the dynamics of the Drosophila populations, the sibling species D. athabasca and D. azteca must be classified unambiguously. We have characterized these two species using three molecular techniques: allozymes, mtDNA and RAPDs. All three techniques allow the classification of any individual as belonging to either species. The study of five localities in northern California and southern Oregon show that the area of overlap is larger than previously described.

Animals↗

PubMed2003

Origin of contamination and genetic diversity of Escherichia coli in beef cattle.

The possible origin of beef contamination and genetic diversity of Escherichia coli populations in beef cattle, on carcasses and ground beef, was examined by using random amplification of polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the fliC gene. E. coli was recovered from the feces of 10 beef cattle during pasture grazing and feedlot finishing and from hides, carcasses, and ground beef after slaughter. The 1,403 E. coli isolates (855 fecal, 320 hide, 153 carcass, and 75 ground beef) were grouped into 121 genetic subtypes by using the RAPD method. Some of the genetic subtypes in cattle feces were also recovered from hides, prechilled carcasses, chilled carcasses, and ground beef. E. coli genetic subtypes were shared among cattle at all sample times, but a number of transient types were unique to individual animals. The genetic diversity of the E. coli population changed over time within individual animals grazing on pasture and in the feedlot. Isolates from one animal (59 fecal, 30 hide, 19 carcass, and 12 ground beef) were characterized by the PCR-RFLP analysis of the fliC gene and were grouped into eight genotypes. There was good agreement between the results obtained with the RAPD and PCR-RFLP techniques. In conclusion, the E. coli contaminating meat can originate from cattle feces, and the E. coli population in beef cattle was highly diverse. Also, genetic subtypes can be shared among animals or can be unique to an animal, and they are constantly changing.

Animals↗

PubMed1999

Typing Listeria monocytogenes by random amplified polymorphic DNA (RAPD) fingerprinting.

Twenty epidemiologically unrelated Listeria monocytogenes strains isolated from different animals, locations and on different dates in Japan were classified into 18 types by the random amplified polymorphic DNA (RAPD) fingerprinting technique with four primers. Further, seven epidemiologically related L. monocytogenes strains isolated from raw milk and a bulk tank on a dairy farm represented the same RAPD type suggesting that they were all of the same origin. Therefore, RAPD-polymerase chain reaction (PCR) analysis, which is rapid, simple and inexpensive to perform, can be used in surveys as a convenient epidemiological technique.

Animals↗

PubMed2003

Presence of yeasts in southern Italian sourdoughs from Triticum aestivum flour.

Samples of sourdoughs obtained from 13 artisanal bakeries located in the Molise and Campania regions were analysed. The sourdoughs were produced with the exclusive use of Triticum aestivum wheat flour. pH values of sourdoughs from Molise were generally lower than those from Campania. The number of yeasts in the samples of sourdoughs from Molise was generally higher than in those from Campania, which in two cases evidenced counts about 2 log cfu x g(-1). By utilising and comparing traditional and biomolecular techniques of identification a complete picture of the isolates was obtained: 58 strains were identified as Saccharomyces cerevisiae, five as Candida colliculosa, four as C. lambica, three as C. krusei, three as C. valida and two as C. glabrata.

Bread↗

PubMed2001

[Advances in molecular biology of dermatophytes].

During the 44th meeting of The Japanese Society for Medical Mycology in Nagasaki, 2000, a forum was held entitled Advances in Molecular Biology of Dermatophytes. Based on the subject, target molecules and kind of approach, we selected seven presentations from over 100 of the poster abstracts. Six of them concerned identification and one concerned viability. Summaries of the 7 presentations are given in this article. Of presentations on the identification methods, 5 demonstrated their usefulness: 1) A sequence analysis of ITS 1 region in ribosomal DNA of several Microsporum species showed ITS 1 genospecies Arthroderma otae to be composed of A. otae, M. canis, M. equinum and M. audouinii. 2) RAPD may be useful for identifying isolates which are not clearly identifiable by conventional biological techniques. 3) Sequence analysis of CHS 1 was shown to be a rapid tool for species level identification of M. gypseum. 4) PCR-SSCP analysis was also useful for discrimination of dermatophytes with high reproducibility and sensitivity. 5) Strain identification of A. benhamiae isolates may be possible using RFLP analysis of NTS regions in ribosomal DNA. The other presentation concerning identification pointed out some important problems: RFLP of mitochondrial DNA and ITS sequencing of A. benhamiae showed that the results are sometimes in conflict with those obtained from biological techniques, or in some cases, between other molecular techniques. This implies that our concept of fungal species needs to be re-examined and perhaps amended. The presentation on viability introduced quantitative analysis of mRNA of ACT gene, a new application of a molecular technique. Since the mRNA expresses only in living cells, the method is highly useful as an indicator of fungal viability.

Actins↗

PubMed2001

High-throughput methods for detection of genetic variation.

Understanding human genetic variation is currently believed to reveal the cause of individual susceptibility to disease and the large variation observed in response to treatment. In this review, we will focus on different approaches to identify and visualize genetic alterations. The various approaches for allele discrimination are formally systematically divided into (i) enzymatic approaches, in which the properties of different enzymes to discriminate between nucleotides are used (restriction enzymes type II, Cleavase and Resolvase, DNA polymerase, and ligase); (ii) electrophoretic methods, in which the allele discrimination is based on the difference in mobility in polymeric gels or capillaries (single- and double-stranded conformation assays, heteroduplex analysis, and DNA sequencing); (iii) solid-phase determination of allelic variants, including high-density oligonucleotide arrays for hybridization analysis, minisequencing primer extension analysis, and fiberoptic DNA sensor array; (iv) chromatographic methods such as denaturing high-performance liquid chromatography (DHPLC); (v) other physical methods of discrimination of allelic variants such as mass spectrometry (mass and charge) or fluorescence exchange-based techniques; and (vi) in silico methods such as high-throughput analysis of expressed sequence tag data. The most frequently used techniques and instrumental settings applied in different combinations are described, and other methods that are less broadly used but have interesting potentials are discussed.

Alleles↗

PubMedDate not supplied

Human but not ovine isolates of Bordetella parapertussis are highly clonal as determined by PCR-based RAPD fingerprinting.

The DNA fingerprints of 170 human isolates and ten ovine isolates of Bordetella parapertussis were examined by arbitrarily-primed PCR/RAPD with 29 primers. Based on this technique, all the human isolates appear highly genetically homogeneous. The ovine isolates could be distinguished from human isolates and they showed diversity among themselves. Therefore, human isolates of B. parapertussis are a highly clonal group adapted to infect humans and they are distinct from polymorphic ovine isolates.

Animals↗

PubMed2006

Distribution of virulence markers in clinical and environmental Vibrio cholerae non-O1/non-O139 strains isolated in Brazil from 1991 to 2000.

One hundred seventy nine Vibrio cholerae non-O1/non-O139 strains from clinical and different environmental sources isolated in Brazil from 1991 to 2000 were serogrouped and screened for the presence of four different virulence factors. The Random Amplification of Polymorphic DNA (RAPD) technique was used to evaluate the genetic relatedness among strains. Fifty-four different serogroups were identified and V. cholerae O26 was the most common (7.8%). PCR analysis for three genes (ctxA, zot, ace) located of the CTX genetic element and one gene (tcpA) located on the VPI pathogenicity island showed that 27 strains harbored one or more of these genes. Eight (4.5%) strains possessed the complete set of CTX element genes and all but one of these belonged to the O26 serogroup suggesting that V. cholerae O26 has the potential to be an epidemic strain. The RAPD profiles revealed a wide variability among strains and no genetic correlation was observed.

Bacterial Proteins↗

PubMedDate not supplied

[DNA from various tissues of the Far Eastern larch and its applicability for RAPD assay].

We studied applicability of RAPD assay for investigating genetic variability of far eastern larches. Techniques to isolate DNA from larch vegetative organs (needles, young shoots and stems) and megagametophytes were selected, and conditions of polymerase chain reaction were optimized. Fifty out of 84 random primers proved efficient, and 22 and 12 primers initiated weak and no synthesis, respectively. The tested primers worked efficiently on the DNA isolated from both diploid and haploid tissues.

DNA, Plant↗

PubMed2004

[Asymmetric somatic hybridization between mixed wheat and Psathyrostachys juncea].

Psathyrostachys juncea is a potential source of useful genes, such as the barley yellow dwarf virus resistance, salt tolerance and drought tolerance, for wheat improvement. Conventional sexual hybridization between wheat and Psathyrostachys juncea is very difficult to occur as the two are sexual incompatible. Somatic hybridization is a promising technique for creating hybrids across the sexual border. Here we report a fusion system for somatic hybridization of wheat using PEG method. Mixed protoplasts of two wheat (Triticum aestivum L. cv. Jinan 177) culture cells (cha9 and 176) were used as the recipients to fuse with the donors, the protoplasts of Psathyrostachys juncea (Fisch.) Nevski irradiated with ultraviolet light (UV) at an intensity of 380 microW/cm2 for 1 min or 2 min. Sixteen clones were generated in the combination I, (wheat 176 + wheat cha9 + P. juncea 1 min UV treatment) and five of the hybrid clones could differentiate to green plants. All the regenerated clones were confirmed as somatic hybrids by cytological, isozyme, chromosome and random amplified polymorphic DNA (RAPD) analysis. Chloroplast genome of the hybrids was analyzed using 7 pairs of wheat-specific chloroplast microsatellite (SSR) primers. Three clones were obtained from the combination II (wheat 176 + wheat cha9 + P. juncea 2 min UV treatment), and all browned slowly and died in 3 months. This result indicated that the mixed wheat cells was helpful to the formation and regeneration of hybrid callus and the dosage of the UV had significant effect on the development of the fusion products.

Chromosomes, Plant↗

PubMed1995

Investigation into the repeated recovery of coagulase-negative staphylococci from blood taken at the end of cardiopulmonary by-pass.

Forty-six strains of coagulase-negative staphylococci (CNS) were typed by biochemical reactivity, antibiotic susceptibility pattern, macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) and arbitrary primed polymerase chain reaction (AP PCR). Twenty-four strains were obtained in 1993 from blood cultures of as many patients coupled to a heart-lung machine during cardiac surgery. Since over 30% of the latter belonged to a single type, it was concluded that during the year of analysis a single clone of CNS persisted in this hospital setting. Subsequent epidemiological surveillance putatively identified four possible carriers among surgical personnel. For this reason, 22 strains collected from the hands and nose of two cardiac surgeons and two perfusionists were also tested; none were identical to the persistent clone. Thus either the operation equipment was colonized longitudinally or the causative CNS had disappeared from the suspect individuals' flora. Longitudinal monitoring of CNS infections by various techniques gives a valuable insight into nosocomial epidemiology and elucidates the complexity of the CNS colonization.

Cardiopulmonary Bypass↗

PubMed2001

Attempt to differentiate Leishmania (Leishmania) amazonensis, L. (L.) chagasi, Leishmania (Viannia) braziliensis and L. (V.) guyanensis using the SSR-PCR technique.

The ability to differentiate reference strains of Leishmania (Leishmania) amazonensis, L. (L.) chagasi, Leishmania (Viannia) braziliensis and L. (V.) guyanensis was evaluated using the simple sequence repeat polymerase chain reaction (SSR-PCR) technique. This technique differentiates the Leishmania species, generating distinct DNA amplicon profiles. The SSR-PCR profiles were similar to but more reproducible than those produced by RAPD. SSR-PCR is presented as an alternative to other molecular methods for the differentiation of Leishmania species or strains.

Animals↗

PubMed1995

DNA fingerprinting of Brassica juncea cultivars using microsatellite probes.

The genetic variability in the Brassica juncea cultivars was detected by employing in-gel hybridization of restricted DNA to simple repetitive sequences such as (GATA)4, (GACA)4 and (CAC)5. The most informative probe/enzyme combination was (GATA)4/EcoRI, yielding highly polymorphic fingerprint patterns for the B. juncea cultivars. This technique was found to be dependable for establishing the variety specific patterns for most of the cultivars studied, a prerequisite for germplasm preservation. The results of the present study were compared with those reported in our earlier study in which random amplification of polymorphic DNA (RAPD) was used for assessing the genetic variability in the B. juncea cultivars.

Base Sequence↗