scholarly journals Development of a Species-Specific PCR-Restriction Fragment Length Polymorphism Analysis Procedure for Identification ofMadurella mycetomatis

1999 ◽  
Vol 37 (10) ◽  
pp. 3175-3178 ◽  
Author(s):  
Abdalla O. A. Ahmed ◽  
Moawia M. Mukhtar ◽  
Marly Kools-Sijmons ◽  
Ahmed H. Fahal ◽  
Sybren de Hoog ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Polymorphism Analysis ◽  
Its Regions ◽  
Species Specific ◽  
Restriction Fragment Length

Madurella mycetomatis is the commonest cause of eumycetoma in Sudan and other countries in tropical Africa. Currently, the early diagnosis of mycetoma is difficult. In attempting to improve the identification of M. mycetomatis and, consequently, the diagnosis of mycetoma, we have developed specific oligonucleotide primers based on the sequence of the internal transcribed spacer (ITS) regions spacing the genes encoding the fungal ribosomal RNAs. The ITS regions were amplified with universal primers and sequenced, and then two sets of species-specific primers were designed which specifically amplify parts of the ITS and the 5.8S ribosomal DNA gene. The new primers were tested for specificity with DNA isolated from human mycetoma lesions and DNA extracted from cultures of M. mycetomatis reference strains and related fungi as well as human DNA. To study the genetic variability of the ITS regions of M. mycetomatis, ITS amplicons were obtained from 25 different clinical isolates and subjected to restriction fragment length polymorphism (RFLP) analysis with CfoI, HaeIII,MspI, Sau3AI, RsaI, andSpeI restriction enzymes. RFLP analysis of the ITS region did not reveal even a single difference, indicating the homogeneity of the isolates analyzed during the current study.

scholarly journals Improved Genotyping Vaccine and Wild-Type Poliovirus Strains by Restriction Fragment Length Polymorphism Analysis: Clinical Diagnostic Implications

2000 ◽  
Vol 38 (12) ◽  
pp. 4337-4342 ◽  
Author(s):  
Amalia Georgopoulou ◽  
Panayotis Markoulatos ◽  
Niki Spyrou ◽  
Nicholas C. Vamvakopoulos
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Clinical Samples ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Restriction Fragment Length

The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5′ noncoding region of polioviruses was selected for RT-PCR amplification by the UC53-UG52 primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, andAvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, orNcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success.

scholarly journals Species-Specific Identification ofMycobacterium leprae by PCR-Restriction Fragment Length Polymorphism Analysis of the hsp65 Gene

1999 ◽  
Vol 37 (6) ◽  
pp. 2016-2019 ◽  
Author(s):  
Nalin Rastogi ◽  
Khye Seng Goh ◽  
Mylene Berchel
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Polymorphism Analysis ◽  
Simple Method ◽  
Hsp65 Gene ◽  
Species Specific ◽  
Restriction Fragment Length

PCR-restriction fragment length polymorphism analysis (PRA) of thehsp65 gene present in all mycobacteria was used in the present investigation to characterize Mycobacterium leprae. Bacilli were extracted and purified from different organs from experimentally infected armadillos and nude mice (Swiss mice ofnu/nu origin). A total of 15 samples were assayed in duplicate, and the results were compared with those obtained for a total of 147 cultivable mycobacteria representing 34 species. Irrespective of its origin or viability, M. leprae strains from all the samples were uniformly characterized by two fragments of 315 and 135 bp upon BstEII digestion and two fragments of 265 and 130 bp upon HaeIII digestion. PRA is a relatively simple method and permits the conclusive identification of M. leprae to the species level.

scholarly journals Species-Specific Identification of Campylobacters by PCR-Restriction Fragment Length Polymorphism and PCR Targeting of the Gyrase B Gene

2008 ◽  
Vol 74 (8) ◽  
pp. 2529-2533 ◽  
Author(s):  
Susumu Kawasaki ◽  
Pina M. Fratamico ◽  
Irene V. Wesley ◽  
Shinichi Kawamoto
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Pcr Assays ◽  
Species Specific ◽  
Pcr Targeting ◽  
Restriction Fragment Length

ABSTRACT PCR-restriction fragment length polymorphism (RFLP) analysis of a 960-bp fragment of the Campylobacter gyrB gene with either DdeI or XspI restriction enzymes generated unique digestion patterns for 12 different Campylobacter species. In addition, PCR assays using species-specific primer sets targeting gyrB were specific for the respective Campylobacter species. Therefore, PCR-RFLP analysis and species-specific PCR assays based on the gyrB gene provide valuable tools for rapid and unambiguous identification of the majority of Campylobacter species.

scholarly journals Restriction Fragment Length Polymorphism Analysis of Anther-culture-derived Solanum chacoense

HortScience ◽  
1994 ◽  
Vol 29 (3) ◽  
pp. 206-208 ◽  
Author(s):  
Ram K. Birhman ◽  
Sylvain R. Rivard ◽  
Mario Cappadocia
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Anther Culture ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Solanum Chacoense ◽  
Polymorphism Analysis ◽  
Restriction Fragment Length

Using restriction fragment length polymorphism (RFLP) analysis, the genetic architecture of some anther-culture-derived S. chacoense Bitt. plants was studied, and their origins were elucidated. Our RFLP analyses showed that 1) several plants, even of different ploidy but otherwise genetically identical (clones), can be regenerated from callus originating from a single microspore and, conversely, that 2) some plants regenerated from single callus can have different genetic constitutions and, therefore, must have originated from two different microspore. These findings imply that previous anther culture efficiency estimates might have to be reconsidered.

scholarly journals Study of Restriction Fragment Length Polymorphism Analysis and Spoligotyping for Epidemiological Investigation ofMycobacterium bovis Infection

1999 ◽  
Vol 37 (10) ◽  
pp. 3217-3222 ◽  
Author(s):  
Eamon Costello ◽  
Donnacha O’Grady ◽  
Orla Flynn ◽  
Rory O’Brien ◽  
Mark Rogers ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Geographic Distribution ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Polymorphism Analysis ◽  
Rflp Type ◽  
Restriction Fragment Length

Restriction fragment length polymorphism (RFLP) analysis with probes derived from the insertion element IS6110, the direct repeat sequence, and the polymorphic GC-rich sequence (PGRS) and a PCR-based typing method called spacer oligonucleotide typing (spoligotyping) were used to strain type Mycobacterium bovis isolates from the Republic of Ireland. Results were assessed for 452 isolates which were obtained from 233 cattle, 173 badgers, 33 deer, 7 pigs, 5 sheep, and 1 goat. Eighty-five strains were identified by RFLP analysis, and 20 strains were identified by spoligotyping. Twenty percent of the isolates were the most prevalent RFLP type, while 52% of the isolates were the most prevalent spoligotype. Both the prevalent RFLP type and the prevalent spoligotype were identified in isolates from all animal species tested and had a wide geographic distribution. Isolates of some RFLP types and some spoligotypes were clustered in regions consisting of groups of adjoining counties. The PGRS probe gave better differentiation of strains than the IS6110 or DR probes. The majority of isolates from all species carried a single IS6110 copy. In four RFLP types IS6110 polymorphism was associated with deletion of fragments equivalent in size to one or two direct variable repeat sequences. The same range and geographic distribution of strains were found for the majority of isolates from cattle, badgers, and deer. This suggests that transmission of infection between these species is a factor in the epidemiology of M. bovis infection in Ireland.

scholarly journals Molecular Fingerprinting of Mycobacterium tuberculosisIsolates Obtained in Havana, Cuba, by IS6110 Restriction Fragment Length Polymorphism Analysis and by the Double-Repetitive-Element PCR Method

1998 ◽  
Vol 36 (10) ◽  
pp. 3099-3102 ◽  
Author(s):  
Ernesto Montoro ◽  
José Valdivia ◽  
Sylvia Cardoso Leão
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Repetitive Element ◽  
Rflp Analysis ◽  
Polymorphism Analysis ◽  
Pcr Method ◽  
Restriction Fragment Length

Mycobacterium tuberculosis sputum isolates from 38 patients, obtained in the first 6 months of 1997 in Havana, Cuba, were characterized by IS6110 restriction fragment length polymorphism (RFLP) analysis and the double-repetitive-element PCR (DRE-PCR) method. Among 41 strains from 38 patients, 24 and 25 unique patterns, and 5 and 4 cluster patterns, were found by the RFLP and DRE-PCR methods, respectively. Patients within two of these clusters were found to be epidemiologically related, while no relation was observed in patients in the other clusters. The DRE-PCR method is rapid, and it was as discriminating as IS6110 RFLP analysis in identifying an epidemiological association. Its simplicity makes the technique accessible for subtyping of M. tuberculosisstrains in laboratories not equipped to perform RFLP analysis.

Sign in / Sign up

Export Citation Format

Share Document