Reverse transcription-polymerase chain reaction (RT-PCR) technique and restriction fragment length polymorphism (RFLP) analysis were used to analyse six isolates of plum pox virus (PPV). Whole coat protein (CP) gene was amplified in four isolates using the unipoty-polyT primer pari. PPV-D was identified by RFLP analysis using SfuI and DraI enzymes in two of the isolates. Two isolates of PPV-M strain yielded RT-PCR products which could not be digested by the two enzymes. Other isolates were subjected to RT-PCR using P1-P2 primers. The specificity of the RT-PCR products was confirmed by AluI digestion, while RsaI digestion enabled strain differentiation. No mixed infection was found.

• MeSH
• Capsid genetics   MeSH
• Plant Diseases virology   MeSH
• Fruit MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Polymorphism, Restriction Fragment Length * MeSH
• RNA, Viral isolation & purification   MeSH
• Rosales virology   MeSH
• Trees virology   MeSH
• Plum Pox Virus classification   genetics   isolation & purification   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Viral MeSH

OBJECTIVES: To characterize by restriction fragment length polymorphism (RFLP) patterns, the distribution of different Mycobacterium tuberculosis strains isolated consecutively from 75 tuberculosis patients who resided in Prague and had culture-confirmed cases during a 4-month period in 1995. METHODS: The insertion sequence IS6110-based RFLP analysis of M. tuberculosis isolates was carried out. RESULTS: There were a total of 75 patients with various forms of tuberculosis (54 males; 21 females). The sources of M. tuberculosis isolates were sputum (n = 64), pleura or lymph node drainage (n = 8), and urine (n = 3). Fifty-three of the patients (70.7%) had isolates with unique RFLP patterns, while 22 (29.3%) had isolates that belonged to seven clusters of related RFLP patterns. The seven clusters consisted of four groups of two patients, two groups of four patients, and one group of six patients. Most of the patients whose isolates fell within a clustered RFLP pattern lived in different quarters of the city and had no identifiable contacts with other patients whose isolates had the same pattern. CONCLUSIONS: The finding that isolates from most patients (70.7%) had unique rather than clustered RFLP patterns suggests that endogenous reactivation rather than exogenous transmission is the major determinant of most of the tuberculosis cases in Prague. The occurrence of seven distinct clusters comprising 29.3% of the isolates suggests that approximately one third of cases developed active tuberculosis from recent exogenous transmission.

• MeSH
• DNA, Bacterial analysis   MeSH
• DNA Fingerprinting MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Lymph Nodes microbiology   MeSH
• Urine microbiology   MeSH
• Molecular Epidemiology MeSH
• Mycobacterium tuberculosis genetics   isolation & purification   MeSH
• Polymorphism, Restriction Fragment Length * MeSH
• Aged MeSH
• Sputum microbiology   MeSH
• Tuberculosis epidemiology   microbiology   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• DNA, Bacterial MeSH

DNA from 1008 strains of Mycobacterium avium subspecies paratuberculosis, digested by restriction endonucleases PstI and BstEII, was hybridised with a standard IS900 probe prepared by PCR and labelled non-radioactively by ECL. DNA fingerprints were scanned by CCD camera and analysed using the software Gel Compar (Applied Maths, Kortrijk, Belgium). Thirteen restriction fragment length polymorphism (RFLP) (PstI) types were detected, which where designated as A, B, C, D, E, F, G, H, I, J, K, L and M in accordance with the study of Pavlik et al. (1995) [Pavlik, I., Bejckova, L., Pavlas, M., Rozsypalova, V., Koskova, S., 1995. Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities. Vet. Microbiol. 45, 311-318]. Twenty RFLP (BstEII) types were detected and designated as C1-3, C5, C7-20, S1 and I1 in accordance with the study by Collins et al. 1990 [Collins, D.M., Gabric, D.M., de Lisle, G.W., 1990. Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization. J. Clin. Microbiol. 28, 1591-1596]. A combination of both RFLP (PstI) and RFLP (BstEII) results revealed a total of 28 different RFLP types. All the RFLP types and detailed protocols are available at Intemet web site WWW...: http:/ /www.vri.cz/wwwrflptext.htm.

• MeSH
• DNA, Bacterial analysis   MeSH
• DNA Fingerprinting standards   MeSH
• DNA Probes MeSH
• Humans MeSH
• Mycobacterium avium subsp. paratuberculosis classification   genetics   isolation & purification   MeSH
• Polymorphism, Restriction Fragment Length MeSH
• Bacterial Typing Techniques standards   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Bacterial MeSH
• DNA Probes MeSH

Several methods of molecular analysis of microbial diversity, including terminal restriction fragment length polymorphism (T-RFLP) analysis are based on measurement of the DNA fragment length. Significant variation between sequence-determined and measured length of restriction fragments (drift) has been observed, which can affect the efficiency of the identification of microorganisms in the analyzed communities. In the past, this variation has been attributed to varying fragment length and purine content. In this study, principal component analysis and multiple regression analysis were applied to find the contributions of those and several other fragment characteristics. We conclude that secondary structure melting point and G+C nucleotide content, besides the fragment length, contribute to the variation observed, whereas the contribution of purine content is less important. Incomplete denaturation of the sample at the start of electrophoretic separation of fragments has been excluded as a major cause of the variation observed. Our regression model explains the observed drift variation by approximately 56%, with standard deviation of the prediction equal to approximately 1.3 bp.

• MeSH
• Amplified Fragment Length Polymorphism Analysis methods   standards   MeSH
• DNA, Fungal chemistry   genetics   MeSH
• Fungi chemistry   genetics   isolation & purification   MeSH
• Nucleic Acid Conformation MeSH
• Phalaris microbiology   MeSH
• Polymorphism, Restriction Fragment Length * MeSH
• Transition Temperature MeSH
• Base Composition MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Fungal MeSH

Restriction fragment length polymorphism analysis of chromosomal DNA from 22 strains of Toxoplasma gondii were characterised using SalI and PstI restriction endonucleases and the TGR1E specific repetitive sequence as a probe. Two virulent strains, RH and P-CZ, had previously been isolated from humans, the remaining 20 strains were isolated from animals in the Czech Republic in 1994 and 1995. Among the 20 recently isolated strains, 19 belonged to an avirulent lineage and only one strain from the wild cat Felis silvestris belonged to a virulent lineage.

• MeSH
• Carnivora MeSH
• Chlorocebus aethiops MeSH
• Fluorescent Antibody Technique, Indirect MeSH
• Genetic Markers MeSH
• Rodentia MeSH
• Cats MeSH
• Rabbits MeSH
• Humans MeSH
• Mice MeSH
• Specific Pathogen-Free Organisms MeSH
• Polymorphism, Restriction Fragment Length * MeSH
• DNA, Protozoan analysis   MeSH
• Dogs MeSH
• Birds MeSH
• Cluster Analysis MeSH
• Toxoplasma genetics   pathogenicity   MeSH
• Vero Cells MeSH
• Virulence MeSH
• Animals MeSH
• Check Tag
• Cats MeSH
• Rabbits MeSH
• Humans MeSH
• Mice MeSH
• Dogs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH
• Names of Substances
• Genetic Markers MeSH
• DNA, Protozoan MeSH

Streptococcus suis is an important pathogen of pigs but is also transmissible to humans, with potentially fatal consequences. Among 29 serotypes currently recognized, some are clinically and epidemiologically more important than others. This is particularly true for serotypes 2 and 14, which have a large impact on pig production and also on human health. Conventional PCR-based serotyping cannot distinguish between serotype 1/2 and serotype 2 or between serotype 1 and serotype 14. Although serotype 1/2 and serotype 2 have a very similar cps locus, they differ in a single-nucleotide substitution at nucleotide position 483 of the cpsK gene. Similarly, serotypes 1 and 14 have a very similar cps locus but also differ in the same nucleotide substitution of the cpsK gene. Fortunately, this cpsK 483G→C/T substitution can be detected by BstNI restriction endonuclease. A PCR-restriction fragment length polymorphism (RFLP) detection method amplifying a fragment of the cpsK gene digested by BstNI restriction endonuclease was developed and tested in reference strains of these serotypes and also in field isolates.

• Keywords
• BstNI, Streptococcus suis, cpsK, diagnostics,
• MeSH
• Polymerase Chain Reaction MeSH
• Polymorphism, Restriction Fragment Length MeSH
• Swine MeSH
• Serogroup MeSH
• Serotyping MeSH
• Streptococcus suis * genetics   MeSH
• Streptococcal Infections * diagnosis   veterinary   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

URA5-RFLP is one of the most widely used genotyping methods relating to Cryptococcus neoformans and C. gattii consensus genotype nomenclature. In order to identify a molecular type, this method uses a visual comparison of digested PCR products of tested and reference strains, therefore any anomaly in RFLP patterns of studied isolates makes recognition difficult or impossible. This report describes a strain of VNIV type showing an atypical URA5-RFLP pattern as well as a group of AD hybrids displaying the same anomaly. The atypical RFLP pattern is the result of a point mutation and emergence of a new restriction site. Emergence of the allele presenting a new banding pattern may lead to misidentification using the URA5-RFLP technique; the results of this study as well as the literature data may suggest the spread of the allele in the environment.

• MeSH
• Cryptococcus neoformans classification   genetics   MeSH
• Genotype MeSH
• Genes, Fungal genetics   MeSH
• Environmental Microbiology MeSH
• Mutation MeSH
• Mycological Typing Techniques MeSH
• Orotate Phosphoribosyltransferase genetics   MeSH
• Polymorphism, Restriction Fragment Length MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Orotate Phosphoribosyltransferase MeSH

A standardised method for PvuII-PstI-IS901 restriction fragment length polymorphism (RFLP) typing was developed and evaluated against 173 isolates of Mycobacterium avium subsp. avium and M. avium subsp. silvaticum originating from birds (N=46) and their aviaries (N=5), pigs (N=85), cattle (N=18), reference serotype strains (N=9), humans (N=7), a horse (N=1), a nutria (N=1), and strain M. avium subsp. avium ST 18 (formerly M. avium subsp. paratuberculosis ST 18). PvuII-IS1245 RFLP typing was also performed on all isolates. DNA was digested in parallel by restriction endonucleases PvuII or PstI and hybridised to standard probes prepared by PCR. DNA fingerprints were scanned by CCD camera and analysed by the Gel Compar (Applied Maths, Version 4.1, Kortrijk, Belgium) software using a standard isolate control profile. A total of 52 PvuII-PstI RFLP profiles was described including 25 PvuII RFLP profiles designated A to Y and 25 PstI RFLP profiles designated A1-L3. Profiles were found to be stable in vivo and in vitro after multiple subcultures. High IS901 copy number was associated with a "bird" PvuII-IS1245 RFLP profile and low IS901 copy number with M. avium subsp. avium isolates from humans and the nutria. A virulence assay of 100 IS901-positive isolates using intramuscular infections of pullets showed 83 isolates differentiated into 32 RFLP types to be virulent and 17 isolates differentiated into 12 RFLP types as nonvirulent. Attenuation of virulence for pullets could be attributed to either multiple in vitro subculture, polyclonal infection or human passage and was not related to IS901 or IS1245 profiles.

• MeSH
• Humans MeSH
• Mycobacterium avium classification   pathogenicity   MeSH
• Polymorphism, Restriction Fragment Length * MeSH
• Birds microbiology   MeSH
• Bacterial Typing Techniques methods   MeSH
• DNA Transposable Elements * MeSH
• Virulence MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Transposable Elements * MeSH

AIMS: The PCR/RFLP typing of 156 isolates Campylobacter jejuni originating from poultry and humans was performed (101 human and 55 poultry strains). METHODS AND RESULTS: On the basis of restrictive digest, six types were identified with AfaI, seven types with MboI and five types with HaeIII. With a combination of these three enzymes, 22 types were found. In human strains, the most frequently occurring types were Cj.4 (28%), Cj.1 (19%), Cj. 13 (13%) and Cj. 2 (5%). In the case of poultry strains, the most frequent types were Cj. 1 (34%), Cj. 11 (22%), C.j. 21 (16%) and Cj. 15 (11%). CONCLUSIONS: The findings support the hypothesis that poultry is a significant source but not sole source of Campylobacter sp. in relation to humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The typing of Campylobacter sp. forms the basis for an evaluation of the current state and risk assessment of various Campylobacter sp. sources in relation to humans.

• MeSH
• Bacteriological Techniques MeSH
• Campylobacter jejuni classification   genetics   MeSH
• Poultry microbiology   MeSH
• Flagellin genetics   MeSH
• Humans MeSH
• Polymerase Chain Reaction * MeSH
• Polymorphism, Restriction Fragment Length * MeSH
• Bacterial Typing Techniques * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Flagellin MeSH

We report that the allele distribution for RFLP's flanking the CF gene differs between patients with and without pancreatic insufficiency. The present study confirms this difference. In both classes the linkage disequilibrium (LD) is highest with the RFLP revealed by probe E9. The haplotype distribution identified by these RFLP's can be used for indirect carrier detection.

• MeSH
• Cystic Fibrosis complications   genetics   MeSH
• Genetic Carrier Screening MeSH
• Exocrine Pancreatic Insufficiency complications   MeSH
• Genetic Markers MeSH
• Haplotypes MeSH
• Humans MeSH
• Molecular Probes MeSH
• Polymorphism, Restriction Fragment Length MeSH
• Linkage Disequilibrium MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Genetic Markers MeSH
• Molecular Probes MeSH