Faculty Opinions recommendation of Rapid identification and differentiation of the soft rot erwinias by 16S-23S intergenic transcribed spacer-PCR and restriction fragment length polymorphism analyses.

2001 ◽  
Author(s):  
Gerard Muyzer
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Soft Rot ◽  
Rapid Identification ◽  
Restriction Fragment Length

scholarly journals Rapid Identification and Differentiation of the Soft Rot Erwinias by 16S-23S Intergenic Transcribed Spacer-PCR and Restriction Fragment Length Polymorphism Analyses

2001 ◽  
Vol 67 (9) ◽  
pp. 4070-4076 ◽  
Author(s):  
I. K. Toth ◽  
A. O. Avrova ◽  
L. J. Hyman
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Soft Rot ◽  
Group Iii ◽  
Group I ◽  
Its Pcr ◽  
Restriction Fragment Length

ABSTRACT Current identification methods for the soft rot erwinias are both imprecise and time-consuming. We have used the 16S-23S rRNA intergenic transcribed spacer (ITS) to aid in their identification. Analysis by ITS-PCR and ITS-restriction fragment length polymorphism was found to be a simple, precise, and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified fromErwinia and other genera using universal PCR primers. After PCR, the banding patterns generated allowed the soft rot erwinias to be differentiated from all other Erwinia and non-Erwinia species and placed into one of three groups (I to III). Group I comprised all Erwinia carotovorasubsp. atroseptica and subsp.betavasculorum isolates. Group II comprised allE. carotovora subsp. carotovora,subsp. odorifera, and subsp. wasabiae andE. cacticida isolates, and group III comprised allE. chrysanthemi isolates. To increase the level of discrimination further, the ITS-PCR products were digested with one of two restriction enzymes. Digestion with CfoI identified E. carotovora subsp.atroseptica and subsp. betavasculorum(group I) and E. chrysanthemi (group III) isolates, while digestion with RsaI identified E. carotovora subsp. wasabiae, subsp. carotovora, and subsp.odorifera/carotovora and E. cacticida isolates (group II). In the latter case, it was necessary to distinguishE. carotovora subsp. odorifera and subsp. carotovora using the α-methyl glucoside test. Sixty suspected soft rot erwinia isolates from Australia were identified as E. carotovora subsp.atroseptica, E. chrysanthemi,E. carotovora subsp. carotovora, and non-soft rot species. Ten “atypical” E. carotovora subsp. atroseptica isolates were identified as E. carotovora subsp.atroseptica, subsp. carotovora, and subsp. betavasculorum and non-soft rot species, and two “atypical” E. carotovora subsp.carotovora isolates were identified as E. carotovora subsp. carotovora and subsp.atroseptica.

Use of PCR-restriction fragment length polymorphism analysis of the hsp65 gene for rapid identification of mycobacteria in Brazil

1999 ◽  
Vol 37 (3) ◽  
pp. 223-229 ◽  
Author(s):  
Adalgiza da Silva Rocha ◽  
Cassiana da Costa Leite ◽  
Hélio Magarinos Torres ◽  
Antonio Basilio de Miranda ◽  
Márcia Quinhones Pires Lopes ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rapid Identification ◽  
Polymorphism Analysis ◽  
Hsp65 Gene ◽  
Restriction Fragment Length

scholarly journals Rapid Identification and Typing ofStaphylococcus aureus by PCR-Restriction Fragment Length Polymorphism Analysis of the aroA Gene

1999 ◽  
Vol 37 (3) ◽  
pp. 570-574 ◽  
Author(s):  
Javier Yugueros Marcos ◽  
Alberto Cascón Soriano ◽  
María Sánchez Salazar ◽  
Carmen Hernanz Moral ◽  
Susana Suárez Ramos ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rapid Identification ◽  
Polymorphism Analysis ◽  
Pcr Products ◽  
Dna Fragment ◽  
Restriction Fragment Length

The Staphylococcus aureus aroA gene, which encodes 5-enolpyruvylshikimate-3-phosphate synthase, was used as a target for the amplification of a 1,153-bp DNA fragment by PCR with a pair of primers of 24 and 19 nucleotides. The PCR products, which were detected by agarose gel electrophoresis, were amplified from all S. aureus strains so far analyzed (reference strains and isolates from cows and sheep with mastitis, as well as 59 isolates from humans involved in four confirmed outbreaks). Hybridization with an internal 536-bp DNA fragment probe was positive for all PCR-positive samples. No PCR products were amplified when other Staphylococcus spp. or genera were analyzed by using the same pair of primers. The detection limit for S. aureus cells was 20 CFU when the cells were suspended in saline; however, the sensitivity of the PCR was lower (5 × 102 CFU) when S. aureus cells were suspended in sterilized whole milk. TaqI digestion of the PCR-generated products rendered two different restriction fragment length polymorphism patterns with the cow and sheep strains tested, and these patterns corresponded to the two different patterns obtained by antibiotic susceptibility tests. Analysis of the 59 human isolates by our easy and rapid protocol rendered results similar to those of other assays.

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