Development of a Brucella suis specific hybridisation probe and PCR which distinguishes B. suis from Brucella abortus.
A genomic library was prepared from Brucella suis DNA (MboI digested) and cloned into the BamHI site of pUC18. Colony hybridisation using a probe prepared from purified B. suis DNA labelled with alpha 32P was carried out to identify colonies of interest. About 20 colonies, which gave an intense signal upon hybridisation with whole B. suis genomic DNA as a probe, were selected. Because of the high degree of DNA homology between B. suis and Brucella abortus, a short probe was chosen as it would more likely give species specificity. Of seven fragments selected to probe whole B. suis, B. abortus, and Yersinia enterocolitica DNA, one was found to hybridise with B. suis only. The probe was sequenced in two directions and sense and anti sense primers of 25bp in length were chosen to yield a product of 421bp. After optimisation of the PCR, a product of 420bp was obtained with B. suis template DNA and two bands of 420 and 650bp were detected with B. abortus template DNA. This is the first reported PCR of the Brucella genome where a single pair of primers will discriminate between B. suis and B. abortus. No band was observed when the two primers were used to amplify E. coli, Y. enterocolitica, Enterobacter cloacae, Staphylococcus aureus, Streptococcus uberis, Corynebacterium bovis, or Serratia marcescens template DNA.
Fayazi
Zahra
Z
Department of Microbiology and Immunology, and the Australian Institute of Tropical Veterinary and Animal Science, James Cook University, Townsville, Qld 4811, Australia.
Ghadersohi
Ali
A
Hirst
Robert G
RG
D000818:Animals; D001483:Base Sequence; D002002:Brucella / Q000235:genetics* / Q000302:isolation & purification; D002003:Brucella abortus / Q000235:genetics* / Q000302:isolation & purification; D002006:Brucellosis / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry; D003937:Diagnosis, Differential; D015698:Genomic Library; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity; D017385:Sequence Homology
D015342:DNA Probes; D004269:DNA, Bacterial