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(Reference retrieved automatically from SciELO through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Fluorescent PCR associated with capillary electrophoresis as a diagnostic tool of bacteria in semen

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Author(s):
Francisca Elda Ferreira Dias ; Cáris Marone Nunes ; Tânia Vasconcelos Cavalcante ; Andréa Azevedo Pires de Castro ; Jorge Luis Ferreira ; José Fernando Garcia
Total Authors: 6
Document type: Journal article
Source: Ciênc. anim. bras.; v. 14, n. 3, p. 366-372, Set. 2013.
Abstract

This study was performed in order to evaluate the detection limit of PCR with fluorescent capillary electrophoresis for Brucella abortus diagnosis in bovine semen. Negative bovine semen samples were artificially contaminated with B. abortus (10(0) to 10(7) bacteria/mL) and DNA was extracted by phenol/chloroform protocol. DNA was amplified by PCR with oligonucleotides previously described BF-5'gcgctcaggctgccgacgcaa3' (6-FAM labeled) and BR-5'accagccattgcggtcggta3' for B. abortus. Oligonucleotides generated DNA fragments of 193 bp. DNA fragments visualization was done under UV light at silver stained 8% poliacrylamide gel, and fluorescent capillary electrophoresis performed in an automatic DNA fragment analyzer. The detection limit of capillary electrophoresis for B. abortus was 10³ bacteria/mL, while for silver stained 8% poliacrylamide gel it was 10(5) bacteria/mL. PCR with fluorescent capillary electrophoresis is fast, efficient and highly sensitive test for DNA detection of Brucella in bovine semen, and itcan be an important tool for health evaluation of the herd and semen sanitary control in artificial insemination centers. (AU)