Development of a multiplex PCR assay for polymorphism analysis of Brucella suis biovars causing brucellosis in swine.
Swine brucellosis is caused by the biovars 1, 2 and 3 of Brucella suis the identification of which up to now relies on microbiological tests lacking adequate specificity together with time consuming and expensive molecular procedures. Based on sequence variation of the omp2b gene, we have developed a four primer set multiplex PCR assay that was tested for polymorphism analysis of B. suis biovars causing brucellosis in swine. The assay exploits the single nucleotide polymorphisms found in omp2b gene of B. suis reference biovars which are conserved in 43 B. suis field isolates from different geographic origins and hosts. Three specific amplification patterns (S1, S2 and S3) were obtained for reference strains of B. suis biovars 1, 2 and 3, respectively. However, some B. suis field isolates identified as biovars 2 or 3 according AMOS-PCR, PCR-RFLP of omp31 and omp2 genes and classical bacteriological methods, resulted also in S1 patterns, limiting the typing usefulness of the method.
Ferrão-Beck
Lénia
L
Laboratório Nacional de Investigação Veterinária, Porto, Portugal. lenia.beck@lniv.min-agricultura.pt
Cardoso
Regina
R
Muñoz
Pilar María
PM
de Miguel
María Jesús
MJ
Albert
David
D
Ferreira
Ana Cristina
AC
Marín
Clara María
CM
Thiébaud
Martine
M
Jacques
Isabelle
I
Grayon
Maggy
M
Zygmunt
Michel S
MS
Garin-Bastuji
Bruno
B
Blasco
Jose María
JM
Sá
Maria Inácia
MI
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D041021:Brucella suis / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D002006:Brucellosis / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D004269:DNA, Bacterial / Q000737:chemistry; D005784:Gene Amplification; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D018272:Porins / Q000737:chemistry / Q000235:genetics*; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000382:microbiology*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C096736:Omp2b protein, Brucella; D018272:Porins