scholarly journals 16S rDNA genotyping using PCR/RFLP (restriction fragment length polymorphism) analysis among the family Vibrionaceae

2006 ◽  
Vol 152 (1) ◽  
pp. 125-132 ◽  
Author(s):  
Hidetoshi Urakawa ◽  
Kumiko Kita-Tsukamoto ◽  
Kouichi Ohwada
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rdna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Polymorphism Analysis ◽  
The Family ◽  
Pcr Rflp ◽  
Restriction Fragment Length

Rapid Estimation of Microbial Populations in Fish Samples by Using Terminal Restriction Fragment Length Polymorphism Analysis of 16S rDNA

2010 ◽  
Vol 73 (1) ◽  
pp. 104-113 ◽  
Author(s):  
YUICHIRO TANAKA ◽  
HAJIME TAKAHASHI ◽  
NAO KITAZAWA ◽  
BON KIMURA
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rdna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Microbial Population ◽  
Fragment Length Polymorphism ◽  
Polymorphism Analysis ◽  
Fish Samples ◽  
Restriction Fragment Length

A rapid system using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting 16S rDNA is described for microbial population analysis in edible fish samples. The defined terminal restriction fragment database was constructed by collecting 102 strains of bacteria representing 53 genera that are associated with fish. Digestion of these 102 strains with two restriction enzymes, HhaI and MspI, formed 54 pattern groups with discrimination to the genus level. This TRFLP system produced results comparable to those from a culture-based method in six natural fish samples with a qualitative correspondence of 71.4 to 92.3%. Using the T-RFLP system allowed an estimation of the microbial population within 7 h. Rapid assay of the microbial population is advantageous for food manufacturers and testing laboratories; moreover, the strategy presented here allows adaptation to specific testing applications.

scholarly journals PCR-Restriction Fragment Length Polymorphism Analysis for Identification of Bacteroides spp. and Characterization of Nitroimidazole Resistance Genes

2000 ◽  
Vol 38 (9) ◽  
pp. 3209-3213 ◽  
Author(s):  
Simon L. J. Stubbs ◽  
Jon S. Brazier ◽  
Paul R. Talbot ◽  
Brian I. Duerden
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rdna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rdna Sequencing ◽  
16S Rdna Pcr ◽  
Pcr Rflp ◽  
Restriction Fragment Length

Bacteroides spp. are opportunist pathogens that cause blood and soft tissue infections and are often resistant to antimicrobial agents. We have developed a combined PCR-restriction fragment length polymorphism (RFLP) technique to characterize the 16S rRNA gene for identification purposes and the nitroimidazole resistance (nim) gene for detection of resistance to the major antimicrobial agent used to treat Bacteroidesinfections: metronidazole (MTZ). PCR-RFLP analysis of 16S ribosomal (rDNA) with HpaII and TaqI produced profiles that enabled discrimination of type strains and identification of 70 test strains to the species level. The 16S rDNA PCR-RFLP identification results agreed with routine phenotypic testing for 62 of the strains. The discrepancies between phenotypic and PCR-RFLP methods for eight strains were resolved by 16S rDNA sequencing in three cases, but five strains remain unidentified. The presence of nim genes was indicated by PCR in 25 of 28 strains that exhibited reduced sensitivity to MTZ. PCR-RFLP of the nim gene products identified the four reported genes (nimA, -B, -C, and -D) and indicated the presence of a previously unreported nim gene in 5 strains. This novelnim gene exhibited 75% DNA sequence similarity withnimB. These rapid, accurate, and inexpensive methods should enable improved identification ofBacteroides spp. and the detection of MTZ resistance determinants.

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