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PubMed1996

Scanning of nucleic acids by in vitro amplification: new developments and applications.

Nucleic acids can be characterized using a variety of "fingerprinting" techniques usually based on nucleic acid hybridization or enzymatic amplification. The scanning of nucleic acids by amplification with arbitrary oligonucleotide primers has become popular because it can generate simple-to-complex patterns from anonymous DNA or RNA templates without requiring prior knowledge of nucleic acid sequence or cloned or characterized probes. Discrete loci are amplified within genomic DNA, DNA complementary to mRNA populations (cDNA), cloned DNA fragments, and even PCR products. The potential and limitations of the various genome scanning techniques, novel improvements, and their recent use in comparative and experimental biology applications, including the analysis of plant and bacterial genomes are discussed.

Alleles↗
PubMed2004

Epidemiology of Burkholderia cepacia complex species recovered from cystic fibrosis patients: issues related to patient segregation.

Studies of the prevalence of Burkholderia cepacia complex species amongst cystic fibrosis (CF) patients in different geographical regions, and the association between cross-infection and putative transmissibility markers, will further our understanding of these organisms and help to address infection-control issues. In this study, B. cepacia complex isolates from CF patients in different regions of Europe were analysed. Isolates were examined for B. cepacia complex species and putative transmissibility markers [cable pilin subunit gene (cblA) and the B. cepacia epidemic strain marker (BCESM)]. Sporadic and cross-infective strains were identified by random amplification of polymorphic DNA (RAPD). In total, 79% of patients were infected with Burkholderia cenocepacia (genomovar III), 18% with Burkholderia multivorans (genomovar II) and less than 5% of patients with B. cepacia (genomovar I), Burkholderia stabilis (genomovar IV) or Burkholderia vietnamiensis (genomovar V). The cblA and BCESM transmissibility markers were only detected in strains of B. cenocepacia. The BCESM was a more sensitive marker for transmissible B. cenocepacia strains than cblA, although sporadic B. cenocepacia strains containing the BCESM, but lacking cblA, were also observed. Furthermore, clusters of cross-infection with transmissibility marker-negative strains of B. multivorans were identified. In conclusion, B. cenocepacia was the greatest cause of cross-infection, and the most widely distributed B. cepacia complex species, within these CF populations. However, cross-infection was not exclusive to B. cenocepacia and cblA and the BCESM were not absolute markers for transmissible B. cenocepacia, or other B. cepacia complex strains. It is therefore suggested that CF centres cohort patients based on the presence or absence of B. cepacia complex infection and not on the basis of transmissibility marker-positive B. cenocepacia as previously suggested.

Bacterial Typing Techniques↗
PubMed2001

Technical note: A quick and more sensitive method to identify pork in processed and unprocessed food by PCR amplification of a new specific DNA fragment.

We developed and evaluated a PCR procedure to detect pork in heated and unheated meat, sausages, canned food, cured products, and pâtés using a faster, more specific, and more sensitive method than others previously described. Isolation of a new DNA-specific porcine repetitive element was performed by nonspecific PCR amplification. After analyzing this repetitive sequence, a pair of primers were synthesized. To confirm the effectiveness and specificity of this fragment, 55 pig blood DNA samples (from differents breeds) were tested and positive results were obtained. With 200 samples tested from other species, the specific pork amplification was not detected. Using this method, we can partially quantify degree of contamination, depending on the PCR amplification cycles, detecting up to 0.005% pork in beef and 1% pork in duck pâté using 30 and 20 PCR amplification cycles, respectively. The amount of porcine DNA detected in cattle DNA was 1.25 and 250 pg when using 30 and 20 amplification cycles, respectively. Pork has been identified in both heated and unheated meat products, sausages, canned food, hamburgers, and pâtés. In conclusion, specific PCR amplification of a repetitive DNA element seems to be a powerful technique for the identification of pork in processed and unprocessed food, because of its simplicity, specificity, and sensitivity (with 30 amplification cycles we can detect 0.005% pork). Furthermore, it is a very fast method, because 1% pork contamination can be detected with 20 PCR cycles. The procedure is also much cheaper than other methods based on RFLP-PCR, immunodiffusion, or other techniques that need expensive equipment.

Animals↗
PubMed1997

[A preliminary study on the identification of crude snake drugs by molecular genetic markers].

DNA was amplified from 11 samples of crude snake drugs by 2 random primers. "Wushaoshe" (Zaocys dhumnades) and its substitutes, Elaphe taeniurus, E. rufodorsata, E. carinata and Sinonatrix annularis, and "Jinqian Baihyashe" (Bungarus multicinctus) and its adulterants, Dinodon rufozonatum and Sinonatrix annularis, can be distinguished by electrophoresis pattern of the amplified products. The results showed that the molecular genetic marker technique can be used as a new method for the identification of crude snake drugs.

Animals↗
PubMed2006

Establishing clonal relationships between VIM-1-like metallo-beta-lactamase-producing Pseudomonas aeruginosa strains from four European countries by multilocus sequence typing.

Ten multidrug-resistant Pseudomonas aeruginosa strains producing VIM-1-like acquired metallo-beta-lactamases (MBLs), isolated from four European countries (Greece, Hungary, Italy, and Sweden), were analyzed for genetic relatedness by several methodologies, including fliC sequence analysis, macrorestriction profiling of genomic DNA by pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), and multilocus sequence typing (MLST). The four approaches yielded consistent results overall but showed different resolution powers in establishing relatedness between isolates (PFGE>RAPD>MLST>fliC typing) and could usefully complement each other to address issues in the molecular epidemiology of P. aeruginosa strains producing acquired MBLs. In particular, the recently developed MLST approach was useful in revealing clonal relatedness between isolates when this was not readily apparent using RAPD and PFGE, and it suggested a common ancestry for some of the VIM-1-like MBL-positive P. aeruginosa strains currently spreading in Europe. The MBL producers belonged in three clonal complexes/burst groups (BGs). Of these, one corresponded to the previously described BG4 and included serotype O12 strains from Hungary and Sweden, while the other two were novel and included serotype O11 or nonserotypable strains from Greece, Sweden, and/or Italy. Comparison of the integrons carrying blaVIM-1-like cassettes of various isolates revealed a remarkable structural heterogeneity, suggesting the possibility that multiple independent events of acquisition of different blaVIM-containing integrons had occurred in members of the same clonal lineage, although a contribution of integrase-mediated cassette shuffling or other recombination mechanisms during the evolution of similar strains could also have played a role in determining this variability.

Bacterial Proteins↗
PubMed1997

Use of random amplification of polymorphic DNA (RAPD) and PCR-fingerprinting for genotyping a Scedosporium prolificans (inflatum) outbreak in four leukemic patients.

Four isolates of the pathogenic fungus Scedosporium prolificans (inflatum), causing a previously reported nosocomial outbreak in four leukemic patients, were typed by random amplification of polymorphic DNA (RAPD) with two different 10-mer primers and PCR-fingerprinting with the core sequence of phage M13 as a single primer. Both techniques allowed 10 additional clinical isolates of Scedosporium prolificans from different areas of Spain, including Scedosporium prolificans NCPF 2884, to be classified into 10 different molecular types. The four outbreak isolates consisted of three molecular types with two patients sharing a similar strain, and the remaining two patients infected by two different strains.

Cross Infection↗
PubMed1998

Comparative study of five different techniques for epidemiological typing of Escherichia coli O157.

A set of 47 Austrian human, food, and veterinary Escherichia coli O157:H7 isolates was used to evaluate five different epidemiological typing methods. Ribotyping using an automated microbial characterization system (RiboPrinter) was not suitable for detection of epidemiological relatedness. All but one E. Coli strain were typeable by phage typing. Random amplified polymorphic DNA-PCR fingerprinting was performed using primer M13 containing the sequence 5'-GAG GGT GGC GGT TCT-3' and primer 1247 (5'-AAGAGCCCGT-3'). Although both methods recognized only two clusters, both dendrograms grouped most of the EHEC O157 isolates into epidemiologically related subgroups. Pulsed-field gel electrophoresis of XbaI digested total DNA was a valuable subtyping system. We found that major differences can exist between results of multiple subtyping methods. E. coli O157 isolates should not be classified as epidemiologically related or nonrelated on the basis of a single typing method alone.

Adolescent↗
PubMed2006

Occurrence, prevalence and genetic environment of CTX-M beta-lactamases in Enterobacteriaceae from Indian hospitals.

OBJECTIVES: To determine occurrence, prevalence and CTX-M genotypes produced by Enterobacteriaceae from clinical samples from three geographically distant Indian hospitals and to detect linkage of IS26 with bla(CTX-M) and map its precise insertion position. METHODS: A total of 130, non-duplicate Escherichia coli and Klebsiella pneumoniae resistant to a third-generation cephalosporin (3GC) from three Indian centres were screened for extended-spectrum beta-lactamase (ESBL) production using phenotypic detection methods. All isolates were screened for bla(CTX-M) using multiplex PCR. Precise CTX-M genotype was identified using reverse-line hybridization. All CTX-M-producing isolates were screened for linkage of IS26 with bla(CTX-M). DNA sequencing was used to map the exact insertion position of this mobile element. RESULTS: Ninety-five of 130 3GC-resistant (73%) (73% of total E. coli, 72% of total K. pneumoniae) isolates were found to carry bla(CTX-M-15). No other CTX-M genotype was detected. IS26 linkage with bla(CTX-M-15) was detected in 31% of isolates carrying bla(CTX-M-15). DNA sequencing revealed variable insertion of this mobile element within tnpA of ISEcp1. RAPD-PCR typing demonstrated great diversity in isolates carrying bla(CTX-M-15); no predominant clone was identified. CONCLUSIONS: In contrast with other studies where greater diversity exists, CTX-M-15 was the only CTX-M ESBL produced in this Indian collection of unrelated E. coli and K. pneumoniae. This is the first systematic survey report from India detecting CTX-M-type beta-lactamases This is also the first report indicating such high mobility/diversity of insertion of IS26 in close association with bla(CTX-M) in a single bacterial collection.

Anti-Bacterial Agents↗
PubMed1996

Molecular epidemiology of Enterobacter aerogenes acquisition: one-year prospective study in two intensive care units.

To evaluate the respective contributions of patient-to-patient transmission and endogenous acquisition of Enterobacter aerogenes isolates, we conducted a prospective epidemiologic study in two intensive care units (ICUs) between May 1994 and April 1995. We collected a total of 185 E. aerogenes isolates: 130 from 51 patients in a surgical ICU (SICU), 45 from 26 patients in a medical ICU (MICU), and 10 from the environments in these two ICUs. All isolates were typed by random amplification of polymorphic DNA and enterobacterial repetitive intergenic consensus PCR. Among the 175 clinical isolate, we observed 40 different profiles by random amplification of polymorphic DNA and 36 different profiles by enterobacterial repetitive intergenic consensus PCR. We identified a ubiquitous and prevalent clone, corresponding to 58% of SICU and 41% of MICU clinical isolates. Three epidemiologically related strains were specific to each ICU and represented 17% of SICU and 24% of MICU clinical isolates; unique type strains represented 17 and 29% of SICU and MICU clinical isolates, respectively, and E. aerogenes strains which were spread to a limited degree and which were isolated less than five times during the 1-year study period represented 8 and 6% of SICU and MICU clinical isolates, respectively. Our results show that E. aerogenes is acquired in the ICU in three different ways: patient-to-patient spread of a prevalent or an epidemiologically related strain, acquisition de novo of a strain from patients' own flora, and acquisition of a nonendemic strain followed by occasional patient-to-patient transmission. The findings point out the importance of patient-to-patient transmission in E. aerogenes acquisition and suggest that changes in E. aerogenes ecology in the hospital have taken place during the past decade.

Adult↗
PubMed2005

Differential gene expression in benznidazole-resistant Trypanosoma cruzi parasites.

We analyzed the differential gene expression among representative Trypanosoma cruzi stocks in relation to benznidazole exposures using a random differentially expressed sequences (RADES) technique. Studies were carried out with drug pressure both at the natural susceptibility level of the wild-type parasite (50% inhibitory concentration for the wild type) and at different resistance levels. The pattern of differential gene expression performed with resistant stocks was compared to the population structure of this parasite, established by random amplified polymorphic DNA analysis and multilocus enzyme electrophoresis. A RADES band polymorphism was observed, and over- or underexpression was linked to the resistance level of the stock. The analysis of RADES bands suggested that different products may be involved in benznidazole resistance mechanisms. No significant association was found between phylogenetic clustering and benznidazole susceptibility. Benznidazole resistance may involve several mechanisms, depending on the level of drug exposure.

Animals↗
PubMed2006

Widespread utility of highly informative AFLP molecular markers across divergent shark species.

Population numbers of many shark species are declining rapidly around the world. Despite the commercial and conservation significance, little is known on even the most fundamental aspects of their population biology. Data collection that relies on direct observation can be logistically challenging with sharks. Consequently, molecular methods are becoming increasingly important to obtain knowledge that is critical for conservation and management. Here we describe an amplified fragment length polymorphism method that can be applied universally to sharks to identify highly informative genome-wide polymorphisms from 12 primer pairs. We demonstrate the value of our method on 15 divergent shark species within the superorder Galeomorphii, including endangered species which are notorious for low levels of genetic diversity. Both the endangered sand tiger shark (Carcharodon taurus, N = 18) and the great white shark (Carcharodon carcharias, N = 7) displayed relatively high levels of allelic diversity. A total of 59 polymorphic loci (H(e) = 0.373) and 78 polymorphic loci (H(e) = 0.316) were resolved in C. taurus and C. carcharias, respectively. Results from other sharks (e.g., Orectolobus ornatus, Orectolobus sp., and Galeocerdo cuvier) produced remarkably high numbers of polymorphic loci (106, 94, and 86, respectively) from a limited sample size of only 2. A major constraint to obtaining much needed genetic data from sharks is the time-consuming process of developing molecular markers. Here we demonstrate the general utility of a technique that provides large numbers of informative loci in sharks.

Animals↗
PubMed1995

[Current developments in Mycobacteria diagnosis].

The reemergence of tuberculosis in the industrialized countries has hastened the development of new laboratory techniques. Hence, well-known shortcomings of traditional techniques such as the lack of a rapid and specific detection system, the delayed availability of species identification and drug susceptibility results, and the lack of a reliable method for determining strain identity for epidemiological purposes, have become immediate targets for implementing molecular biology techniques. In particular, nucleic acid amplification techniques, restriction fragment-length polymorphism and single-strand conformation polymorphism analyses have dramatically improved diagnostic timeliness and accuracy of mycobacteriology laboratory results. Our paper reviews recent developments and comments on the diagnostic applications of the new tools as compared to traditional techniques.

Amino Acid Sequence↗
PubMed2002

Multicenter comparison of three different analytical systems for evaluation of DNA banding patterns from Cryptococcus neoformans.

The enormous improvement of molecular typing techniques for epidemiological and clinical studies has not always been matched by an equivalent effort in applying optimal criteria for the analysis of both phenotypic and molecular data. In spite of the availability of a large collection of statistical and phylogenetic methods, the vast majority of commercial packages are limited by using only the unweighted pair group method with arithmetic mean algorithm to construct trees and by considering electrophoretic pattern only as migration distances. The latter method has serious drawbacks when different runs (separate gels) of the same molecular analysis are to be compared. This work presents a multicenter comparison of three different systems of banding pattern analysis on random amplified polymorphic DNA, (GACA)(4), and contour-clamped homogeneous electric field patterns from strains of Cryptococcus neoformans var. neoformans isolated in different clinical and geographical situations and a standard Saccharomyces cerevisiae strain employed as an outgroup. The systems considered were evaluated for their actual ability to(i) recognize identities, (ii) define complete differences (i.e., the ability to place S. cerevisiae out of the C. neoformans cluster), and (iii) estimate the extent of similarity among different strains. The ability to cluster strains according to the patient from which they were isolated was also evaluated. The results indicate that different algorithms do indeed produce divergent trees, both in overall topology and in clustering of individual strains, thus suggesting that care must be taken by individual investigators to use the most appropriate procedure and by the scientific community in defining a consensus system.

Algorithms↗
PubMed2004

Bradyrhizobium betae sp. nov., isolated from roots of Beta vulgaris affected by tumour-like deformations.

Some varieties of sugar beet, Beta vulgaris, cultivated in northern Spain have large deformations that resemble the tumours produced by Agrobacterium species. In an attempt to isolate the agent responsible for these deformations, several endophytic slow-growing bacterial strains were isolated, the macroscopic morphology of which resembled that of Bradyrhizobium species. These strains were not able to produce tumours in Nicotiana tabacum plants and, based on phylogenetic analysis of their 16S rRNA, they are closely related to the genus Bradyrhizobium. Phenotypic and molecular characteristics of these strains revealed that they represent a species different from all Bradyrhizobium species previously described. Sequence analysis of the 16S-23S rDNA intergenic spacer region indicated that these novel strains form a homogeneous group, related to Bradyrhizobium japonicum, Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense. DNA-DNA hybridization confirmed that these strains represent a novel species of the genus Bradyrhizobium, for which the name Bradyrhizobium betae sp. nov. is proposed. The type strain is PL7HG1T (=LMG 21987T=CECT 5829T).

Bacterial Typing Techniques↗
PubMed1996

Comparative typing of Pseudomonas aeruginosa by random amplification of polymorphic DNA or pulsed-field gel electrophoresis of DNA macrorestriction fragments.

Eighty-seven strains of Pseudomonas aeruginosa were typed by random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments. Stains were clustered on the basis of interpretative criteria as presented previously for the PFGE analysis. Clusters of strains were also defined on the basis of epidemiological data and subsequently reanalyzed by RAPD. It was found that in an RAPD assay employing the enterobacterial repetitive intergenic consensus sequence ERIC2 as a primer, single band differences can be ignored; in this case, clonally related strains could be grouped as effectively and reliably as with PFGE. These data could be corroborated by the use of other primer species. However, some primers either showed reduced resolution or, in contrast, identified DNA polymorphisms beyond epidemiologically and PFGE-defined limits. Apparently, different primers define different windows of genetic variation. It is suggested that criteria for interpretation of the ERIC2 PCR fingerprints can be simple and straightforward: when single band differences are ignored, RAPD-determined grouping of P. aeruginosa is congruent with that obtained by PFGE. Consequently, this implies that RAPD can be used with trust as a first screen in epidemiological characterization of P. aeruginosa. The ability to measure the rate of molecular evolution of the P. aeruginosa genome clearly depends on the choice of restriction enzyme or primer when RAPD or PFGE, respectively, is applied for the detection of DNA polymorphisms.

Bacterial Typing Techniques↗
PubMed1997

Identification of pathogenic Leptospira genospecies by continuous monitoring of fluorogenic hybridization probes during rapid-cycle PCR.

Partial sequences of 23S rRNA gene PCR products from 23 strains of 6 pathogenic Leptospira genospecies and from 8 strains of the saprophytic Leptospira biflexa were determined. Sequence analyses enabled Leptospira genus-specific amplification primers and pathogen-specific fluorogenic adjacent hybridization probes to be designed and synthesized. A PCR protocol was developed in which changes in fluorescence emission resulting from specific annealing of fluorogenic adjacent hybridization probes to the target DNA were continuously monitored. Nine strains of the pathogenic Leptospira genospecies could be differentiated from Leptonema illini, Escherichia coli, and eight strains of Leptospira biflexa. The PCR method was rapid, requiring 18 min for the completion of 45 cycles. It was also simple and flexible, as DNA templates prepared by four different methods, including the simple boiling method, could be used without adverse effects. Two hundred copies of target, equivalent to 100 cells, could be detected.

Animals↗
PubMed2001

Rep-PCR--a variant to RAPD or an independent technique of bacteria genotyping? A comparison of the typing properties of rep-PCR with other recognised methods of genotyping of microorganisms.

The paper presents technical aspects of rep-PCR fingerprinting technique and compares its typing abilities, differentiation power and reproducibility with other recognised and recommended genotyping methods. Although rep-PCR fingerprinting is similar to MAAP techniques, it demonstrates some essentially different elements. The data presented in this review, indicate a rep-PCR genomic fingerprinting technique as a highly discriminating, independent screening method for determining the taxonomical diversity of bacterial population.

Bacteria↗