Validation of the multiplex PCR for identification of Brucella spp.

Orzil, Lívia de Lima; Preis, Ingred Sales; Almeida, Iassudara Garcia de; Souza, Patrícia Gomes de; Soares Filho, Paulo Martins; Jacinto, Fabrício Barcelos; Fonseca Júnior, Antônio Augusto

doi:10.1590/0103-8478cr20150065

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MICROBIOLOGY • Cienc. Rural 46 (05) • May 2016 • https://doi.org/10.1590/0103-8478cr20150065 copy

Validation of the multiplex PCR for identification of Brucella spp.

Validação da técnica de PCR multiplex para identificação de Brucella spp.

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT:

A multiplex PCR technique for detection of Brucella spp. in samples of bacterial suspension was validated as a complementary tool in the diagnosis of the disease. This technique allows the characterization of the agent without performing biochemical tests, which greatly reduces the time for a final diagnosis, and provides more security for the analyst by reducing the time of exposure to microorganisms. The validation was performed in accordance with the Manual of Diagnostic Tests from OIE (2008) and following the requirements present in the ABNT NBR ISO/IEC 17025:2005. The mPCR validated in this study identified the different species ofBrucella (Brucella abortus , B. suis ,B. ovis eB. melitensis ) of bacterial suspension obtained from the slaughterhouse samples, as well as distinguished the biovars (1, 2 e 4; 3b, 5, 6 e 9) ofB. abortus in grouped form and differentiated the field strains from vaccine strains, as a quick, useful and less expensive technique in diagnosis of brucellosis in Brazil.

Key words:
Brucellosis; molecular identification; zoonosis; Brucella ; multiplex PCR

Authorship

Lívia de Lima Orzil

Ministério da Agricultura, Pecuária e Abastecimento (MAPA), Pedro Leopoldo, MG, Brasil. Ministério da Agricultura, Pecuária e AbastecimentoBrazilPedro Leopoldo, MG, BrazilMinistério da Agricultura, Pecuária e Abastecimento (MAPA), Pedro Leopoldo, MG, Brasil.

Ingred Sales Preis

Iassudara Garcia de Almeida

Patrícia Gomes de Souza

Paulo Martins Soares Filho

Fabrício Barcelos Jacinto

Serviço de Inspeção Federal, Ministério da Agricultura, Pecuária e Abastecimento (MAPA), Pedro Leopoldo, MG, Brasil.Ministério da Agricultura, Pecuária e AbastecimentoBrazilPedro Leopoldo, MG, BrazilServiço de Inspeção Federal, Ministério da Agricultura, Pecuária e Abastecimento (MAPA), Pedro Leopoldo, MG, Brasil.

Antônio Augusto Fonseca Júnior^**Corresponding author: Antônio Augusto Fonseca Júnior, email: antonio.a.fonsecajr@gmail.com.

^*Corresponding author: Antônio Augusto Fonseca Júnior, email: antonio.a.fonsecajr@gmail.com.

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (3)
Tables (3)

Thumbnail

Figure 1
1.5% agarose gel electrophoresis showing the different patterns for each species and biovars of B . abortus using the GoTaq enzyme. Markers at intervals of 100 base pairs. 1 to 7: B. abortus , samples used in repeatability test. 8: B. abortus biovar 1. This same profile is observed to B. abortus biovars 2 and 4; 9: B. abortus S19. 10: B. abortus biovar 1; 11: B. abortus biovar 3b. This same profile is observed to B. abortus biovars 5, 6 and 9; 12: B. melitensis and 13: B. suis , 14: blank control (No DNA template). 15: MM (Molecular Marker).

Thumbnail

Figure 2
1.5% agarose gel electrophoresis demonstrating the results after the mPCR was performed with the Jump Start enzyme (final concentration 0.1U uL^-1). Markers at intervals of 100 base pairs. In this robustness test, we did not observe the pattern of bands expected using this enzyme, and is therefore unsuitable for this reaction. Samples: 1 - DB 012/12 23 T1; 2 - DB 012/12 16 T1; 3 - DB 012/12 1 T1; 4 - DB 012/12 2 T1; 5 - DB 012/12 25 TI; 6 - DB 012/12 41 T1; 7 - DB 012/12 26 T1; 8 - B. abortus biovar 1. This same profile is observed to B . abortus biovars 2 and 4; 9 - B . abortus S19; 10 - B . abortus biovar 1; 11 - B . abortus biovar 3b. This same profile is observed to B. abortus biovars 5, 6 and 9; 12 - B. melitensis ; 13 - B. suis ; 14 - Blank (No DNA template); 15 - MM (Molecular Marker).

Thumbnail

Figure 3
1.5% agarose gel electrophoresis demonstrating the results after the mPCR, which was performed with the Taq Platinum enzyme (final concentration of 0.05U uL^-1). Just as was observed with the Jump Start enzyme, we did not observed the pattern of bands expected in this reaction. Samples: 1 - DB 012/12 23 T1; 2 - DB 012/12 16 T1; 3 - DB 12/12 1 T1; 4 - DB 12/12 2 T1; 5 - DB 12/12 25 T1; 6 - DB 12/12 41 T1; 7 - DB 12/12 26 T1; 8 - B. abortus biovar 1. This same profile is observed to B. abortus biovars 2 and 4; 9 - B. abortus S19; 10 - B. abortus biovar 1; 11 - B. abortus biovar 3b. This same profile is observed to B. abortus biovars 5, 6 and 9; 12 - B. melitensis ; 13 - B. suis ; 14 - Blank (No DNA template); 15 - MM (Molecular Marker).

Thumbnail

Table 1
DNA sequence of the primers used in the validation of the mPCR, gene target and reference.

Thumbnail

Table 2
Electrophoresis profiles of Brucella spp. observed according to the primers used on mPCR.

Thumbnail

Table 3
List of biovars of Brucella abortus identified in bacterial isolation and their Brazilian State.

Figure 1 1.5% agarose gel electrophoresis showing the different patterns for each species and biovars of B . abortus using the GoTaq enzyme. Markers at intervals of 100 base pairs. 1 to 7: B. abortus , samples used in repeatability test. 8: B. abortus biovar 1. This same profile is observed to B. abortus biovars 2 and 4; 9: B. abortus S19. 10: B. abortus biovar 1; 11: B. abortus biovar 3b. This same profile is observed to B. abortus biovars 5, 6 and 9; 12: B. melitensis and 13: B. suis , 14: blank control (No DNA template). 15: MM (Molecular Marker).

Figure 2 1.5% agarose gel electrophoresis demonstrating the results after the mPCR was performed with the Jump Start enzyme (final concentration 0.1U uL^-1). Markers at intervals of 100 base pairs. In this robustness test, we did not observe the pattern of bands expected using this enzyme, and is therefore unsuitable for this reaction. Samples: 1 - DB 012/12 23 T1; 2 - DB 012/12 16 T1; 3 - DB 012/12 1 T1; 4 - DB 012/12 2 T1; 5 - DB 012/12 25 TI; 6 - DB 012/12 41 T1; 7 - DB 012/12 26 T1; 8 - B. abortus biovar 1. This same profile is observed to B . abortus biovars 2 and 4; 9 - B . abortus S19; 10 - B . abortus biovar 1; 11 - B . abortus biovar 3b. This same profile is observed to B. abortus biovars 5, 6 and 9; 12 - B. melitensis ; 13 - B. suis ; 14 - Blank (No DNA template); 15 - MM (Molecular Marker).

Figure 3 1.5% agarose gel electrophoresis demonstrating the results after the mPCR, which was performed with the Taq Platinum enzyme (final concentration of 0.05U uL^-1). Just as was observed with the Jump Start enzyme, we did not observed the pattern of bands expected in this reaction. Samples: 1 - DB 012/12 23 T1; 2 - DB 012/12 16 T1; 3 - DB 12/12 1 T1; 4 - DB 12/12 2 T1; 5 - DB 12/12 25 T1; 6 - DB 12/12 41 T1; 7 - DB 12/12 26 T1; 8 - B. abortus biovar 1. This same profile is observed to B. abortus biovars 2 and 4; 9 - B. abortus S19; 10 - B. abortus biovar 1; 11 - B. abortus biovar 3b. This same profile is observed to B. abortus biovars 5, 6 and 9; 12 - B. melitensis ; 13 - B. suis ; 14 - Blank (No DNA template); 15 - MM (Molecular Marker).

Table 1 DNA sequence of the primers used in the validation of the mPCR, gene target and reference.

Table 2 Electrophoresis profiles of Brucella spp. observed according to the primers used on mPCR.

Table 3 List of biovars of Brucella abortus identified in bacterial isolation and their Brazilian State.

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Universidade Federal de Santa Maria Universidade Federal de Santa Maria, Centro de Ciências Rurais , 97105-900 Santa Maria RS Brazil , Tel.: +55 55 3220-8698 , Fax: +55 55 3220-8695 - Santa Maria - RS - Brazil
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[1] ^*Corresponding author: Antônio Augusto Fonseca Júnior, email: antonio.a.fonsecajr@gmail.com.