Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes

Appl Environ Microbiol. 1996 May;62(5):1683-8. doi: 10.1128/aem.62.5.1683-1688.1996.

Abstract

The 16S-23S rRNA spacer regions of Brucella abortus, B. melitensis, and B. suis were cloned and subcloned after PCR amplification. Sequence analysis of the inserts revealed a spacer of about 800 bp with very high ( > 99%) homology among the three species examined. Two genus-specific primer pairs, BRU-P5-BRU-P8 and BRU-P6-BRU-P7, that could be used in a nested PCR format and three genus-specific DNA probes, BRU-ICG2, BRU-ICG3, and BRU-ICG4, were deduced from this spacer. The specificity and sensitivity of both primer sets and probes were examined by testing them against a collection of 18 Brucella strains and 56 strains from other relevant taxa by using PCR and the Line Probe Assay (LiPA), respectively. A method for direct detection of Brucella spp. in 1 ml of raw milk was developed on the basis of enzymatic treatment of the milk components and subsequent PCR and LiPA hybridization. After a single PCR, sensitivities of 2.8 x 10(5) and 2.8 x 10(4) CFU/ml were obtained for detection by agarose gel electrophoresis and LiPA, respectively. Nested PCR yielded a sensitivity of 2.8 x 10(2) CFU/ml for both methods.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Brucella / genetics
  • Brucella / isolation & purification*
  • Cattle
  • DNA Probes
  • Food Microbiology*
  • Milk / microbiology*
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • RNA, Bacterial / analysis*
  • RNA, Ribosomal, 16S / analysis*
  • RNA, Ribosomal, 23S / analysis*

Substances

  • DNA Probes
  • RNA, Bacterial
  • RNA, Ribosomal, 16S
  • RNA, Ribosomal, 23S

Associated data

  • GENBANK/X95889
  • GENBANK/X95890
  • GENBANK/X95891
  • GENBANK/X95892