scholarly journals Identification protocol for sixArmillariaspecies from northeastern North America

2010 ◽  
Vol 40 (3) ◽  
pp. 536-548 ◽  
Author(s):  
J. A. McLaughlin ◽  
T. Hsiang
Keyword(s):  
Dna Sequences ◽  
Fragment Length Polymorphism ◽  
Intergenic Spacer ◽  
Polymorphism Analysis ◽  
Specific Primers ◽  
Northeastern North America ◽  
Armillaria Ostoyae ◽  
Armillaria Gallica ◽  
Species Specific ◽  
Restriction Fragment Length

DNA sequences (~3 kb long) extending from the intergenic spacer 1 (IGS1) region to the 18S gene were obtained for isolates of Armillaria ostoyae , Armillaria calvescens , Armillaria gallica , and Armillaria sinapina . Additional investigation of 16 A. ostoyae, 11 Armillaria gemina , 21 A. calvescens, 18 A. gallica, and 15 A. sinapina isolates produced 117 sequences spanning the 3′ end of the IGS1 through the 5S gene and into the 5′ end of the IGS2 region. Additional sequences spanning the 3′ IGS2 to 5′ 18S gene region were obtained for two A. ostoyae, three A. gemina, two A. calvescens, two A. gallica, and three A. sinapina isolates. This is the first report of complete IGS2 sequences from Armillaria spp. A species identification protocol involving species-specific primers and restriction fragment length polymorphism analysis was devised based on species-specific polymorphisms. The protocol successfully identified all 16 A. ostoyae, 11 A. gemina, three of three Armillaria mellea , 18 A. gallica, 14 of 15 A. sinapina (11/12 diploid and 3/3 haploid), and 14 of 21 A. calvescens (13/15 diploid and 1/6 haploid) isolates included in this study. To the best of our knowledge, this success rate has not been matched by other methods.

scholarly journals Identification of Carnobacterium Species by Restriction Fragment Length Polymorphism of the 16S-23S rRNA Gene Intergenic Spacer Region and Species-Specific PCR

2004 ◽  
Vol 70 (8) ◽  
pp. 4468-4477 ◽  
Author(s):  
Cinta Rachman ◽  
Petia Kabadjova ◽  
Rosica Valcheva ◽  
Hervé Prévost ◽  
Xavier Dousset
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Intergenic Spacer ◽  
Specific Primers ◽  
Intergenic Spacer Region ◽  
Pcr Rflp ◽  
Species Specific ◽  
Restriction Fragment Length

ABSTRACT The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species. Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for all Carnobacterium species. The L-ISR sequence revealed the presence of two tRNA genes, tRNAAla and tRNAIle, which were separated by a spacer region that varied from 24 to 38 bp long. This region was variable among the species, allowing the design of species-specific primers. These primers were tested and proved to be species specific. The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of the Carnobacterium species from other phylogenetically related lactic acid bacteria.

scholarly journals Identification of Porphyromonas gingivalis Strains by Heteroduplex Analysis and Detection of Multiple Strains

1999 ◽  
Vol 37 (12) ◽  
pp. 3906-3911 ◽  
Author(s):  
Eugene J. Leys ◽  
James H. Smith ◽  
Sharon R. Lyons ◽  
Ann L. Griffen
Keyword(s):  
Porphyromonas Gingivalis ◽  
Dna Sequences ◽  
Allelic Variation ◽  
Intergenic Spacer ◽  
Heteroduplex Analysis ◽  
Clinical Samples ◽  
Specific Primers ◽  
Intergenic Spacer Region ◽  
Species Specific ◽  
Multiple Strains

Heteroduplex analysis has been used extensively to identify allelic variation among mammalian genes. It provides a rapid and reliable method for determining and cataloging minor differences between two closely related DNA sequences. We have adapted this technique to distinguish among strains or clonal types of Porphyromonas gingivalis. The ribosomal intergenic spacer region (ISR) was amplified directly from a subgingival plaque sample by PCR with species-specific primers, avoiding the need for culturing the bacteria. The PCR products were then directly compared by heteroduplex analysis with known strains of P. gingivalis for identification. We identified 22 distinct but closely related heteroduplex types ofP. gingivalis in 1,183 clinical samples. Multiple strains were found in 34% of the samples in which P. gingivaliswas detected. Heteroduplex types were identified from these multistrain samples without separating them by culturing or molecular cloning. PCR with species-specific primers and heteroduplex analysis makes it possible to reliably and sensitively detect and identify strains ofP. gingivalis in large numbers of samples.

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 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.

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