title: Evaluation of DNA extraction protocols for Brucella abortus pcr detection in aborted fetuses or calves born from cows experimentally infected with strain 2308.
abstract: The objective of the present study was to improve the detection of B. abortus by PCR in organs of aborted fetuses from infected cows, an important mechanism to find infected herds on the eradication phase of the program. So, different DNA extraction protocols were compared, focusing the PCR detection of B. abortus in clinical samples collected from aborted fetuses or calves born from cows challenged with the 2308 B. abortus strain. Therefore, two gold standard groups were built based on classical bacteriology, formed from: 32 lungs (17 positives), 26 spleens (11 positives), 23 livers (8 positives) and 22 bronchial lymph nodes (7 positives). All samples were submitted to three DNA extraction protocols, followed by the same amplification process with the primers B4 and B5. From the accumulated results for organ, the proportion of positives for the lungs was higher than the livers (p=0.04) or bronchial lymph nodes (p=0.004) and equal to the spleens (p=0.18). From the accumulated results for DNA extraction protocol, the proportion of positives for the Boom protocol was bigger than the PK (p< 0.0001) and GT (p=0.0004). There was no difference between the PK and GT protocols (p=0.5). Some positive samples from the classical bacteriology were negative to the PCR and vice-versa. Therefore, the best strategy for B. abortus detection in the organs of aborted fetuses or calves born from infected cows is the use, in parallel, of isolation by classical bacteriology and the PCR, with the DNA extraction performed by the Boom protocol.
authors.affiliation: Departamento de Medicina Veteriná ria Preventiva e Saúde Animal, Faculdade de Medicina Veteriná ria e Zootecnia, Universidade de São Paulo , São Paulo, SP , Brasil.
authors.forename: M
authors.initials: M
authors.lastname: Matrone
authors.forename: L B
authors.initials: LB
authors.lastname: Keid
authors.forename: V C M
authors.initials: VC
authors.lastname: Rocha
authors.forename: M P
authors.initials: MP
authors.lastname: Vejarano
authors.forename: C Y
authors.initials: CY
authors.lastname: Ikuta
authors.forename: C A R
authors.initials: CA
authors.lastname: Rodriguez
authors.forename: F
authors.initials: F
authors.lastname: Ferreira
authors.forename: R A
authors.initials: RA
authors.lastname: Dias
authors.forename: J S
authors.initials: JS
authors.lastname: Ferreira Neto
keywords: DNA extraction; PCR; abortion; bovine; brucellosis
references.citation: Biotechniques. 1993 Sep;15(3):532-4, 536-7
references.pmid: 7692896
references.citation: Vaccine. 2006 Jun 19;24(25):5327-34
references.pmid: 16713034
references.citation: Vet Microbiol. 2002 Dec 20;90(1-4):435-46
references.pmid: 12414163
references.citation: Eur J Clin Microbiol Infect Dis. 2001 Feb;20(2):127-31
references.pmid: 11305467
references.citation: J Clin Microbiol. 1995 Dec;33(12):3087-90
references.pmid: 8586678
references.citation: J Clin Microbiol. 2001 Apr;39(4):1661-4
references.pmid: 11283112
references.citation: J Clin Microbiol. 1996 Feb;34(2):477-8
references.pmid: 8789045
references.citation: Clin Infect Dis. 1992 Apr;14(4):902-7
references.pmid: 1576286
references.citation: J Vet Diagn Invest. 1992 Jan;4(1):79-83
references.pmid: 1554774
references.citation: Appl Environ Microbiol. 1997 Oct;63(10):3741-51
references.pmid: 9327537
references.citation: J Trop Med Hyg. 1992 Aug;95(4):271-5
references.pmid: 1495123
references.citation: J Clin Microbiol. 1990 Mar;28(3):495-503
references.pmid: 1691208
references.citation: Vet Microbiol. 2003 May 2;93(1):53-61
references.pmid: 12591206
references.citation: Vet Microbiol. 2002 Jun 20;87(2):139-47
references.pmid: 12034542
references.citation: Aust Vet J. 2001 Jul;79(7):500-1
references.pmid: 11549051
references.citation: Clin Lab. 2003;49(9-10):487-505
references.pmid: 14572205
references.citation: Infez Med. 1999;7(2):119-124
references.pmid: 12759592
references.citation: J Appl Bacteriol. 1990 Aug;69(2):216-27
references.pmid: 2272943
references.citation: Clin Infect Dis. 1992 Dec;15(6):1068-9
references.pmid: 1457654