[논문]DNA fingerprinting of Brucella abortus isolated from bovine brucellosis outbreaks by repetitive element sequence (rep)-PCR

Suh, Dong Kyun

DNA fingerprinting of Brucella abortus isolated from bovine brucellosis outbreaks by repetitive element sequence (rep)-PCR 원문보기

大韓獸醫學會誌 = Korean journal of veterinary research, v.45 no.2 = no.119, 2005년, pp.199 - 205

Suh, Dong Kyun (Research Institute of Health and Environment)

Abstract ▼ AI-Helper

DNA fingerprint patterns of 8 Brucella reference strains and 15 B. abortus field isolates were characterized by repetitive element sequence-based PCR (rep-PCR) using BOX- and ERIC-primers in this study. AMOS PCR differentiated all Brucella field isolates from B. abortus RB51, a vaccine strain by producing a B. abortus-specific 498 bp band. Rep-PCR using BOX-primer produced 13 to 18 bands with sizes of between 230 and 3,300 bp, and discriminated Brucella strains to the species level except B. canis and B. suis. PCR products amplified with ERIC primers were, however, not appropriate for differentiating the Brucella isolates. DNA fingerprint patterns for all B. abortus field isolates were identical among them and were put on one cluster with B. abortus biovar 1 reference strain in the dendrogram, indicating they were highly clonal. These results suggested that rep-PCR using BOX primer might to be a useful tool for calculating genetic relatedness among the Brucella species and for the study of brucellosis epidemiology.

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제안 방법

Isolates were tested by the Brucella AMOS (acronym for B. abortus, melitensis, ovis and suis) PCR to identify B. abortus isolates and differentiate them from B. abortus RB51, a vaccine strain [10]. PCR was performed as previously described [10].
The AMOS PCR was performed to confirm the identification of B. abortus in this study. It was based on the fact that the genetic element IS711 occurs at several species-specific (or biovar-specific) chromosomal loci.
It was based on the fact that the genetic element IS711 occurs at several species-specific (or biovar-specific) chromosomal loci. The assay was designed to amplify speciesspecific- sized products by using five primers, one of which hybridizes to one of four species-specific regions adjacent to the element. Thus, the products were composed of a portion of the IS711 element and a predetermined number of nucleotides flanking the 3 end of the element at a species-specific locus [5].

대상 데이터

Samples were collected from bovine brucellosis outbreaks on the course of slaughtering due to positive serum tube test. The outbreaks occurred from two different regions and from different sources of dairy and Korean native cattle between 2001 and 2003. Bovine tissues were cultured on Brucella agar (Difco, USA) supplemented with antibiotics, and incubated at 37oC in an atmosphere of 10 percent carbon according to the methods and criteria described by Alton et al.

이론/모형

A dendrogram was constructed with Analysis software (Biometra, Germany). The patterns were compared by means of the Dice coefficient of band-based similarity by unweighted pair group method using averages (UPGMA); a tolerance of 5% in the band position was applied.

성능/효과

abortus biovar 1. Also, the fact that all field isolates showed a different fingerprint pattern with B. abortus RB51, a vaccine strain, demonstrated that the outbreaks were not caused by a vaccine strain.
Thus, the products were composed of a portion of the IS711 element and a predetermined number of nucleotides flanking the 3 end of the element at a species-specific locus [5]. The PCR result was in 100% agreement with the conventional biochemical identification procedures in identifying the B. abortus field isolates tested. Previous study [5], however, reported that B.
We used ERIC- and BOX primers to distinguish Brucella strains that belong to different species and those isolated from bovine brucellosis outbreaks. The results in this study showed that BOX-PCR provided the distinct patterns for Brucella species. All Brucella reference strains tested could be discriminated at least to the species level except B.

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