Rijpens N P, Jannes G, Van Asbroeck M, Rossau R, Herman L M
Government Dairy Research Station, Melle, Belgium.
Appl Environ Microbiol. 1996 May;62(5):1683-8. doi: 10.1128/aem.62.5.1683-1688.1996.
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.
流产布鲁氏菌、羊种布鲁氏菌和猪种布鲁氏菌的16S - 23S rRNA间隔区经PCR扩增后进行克隆和亚克隆。对插入片段的序列分析显示，在所检测的三个菌种中，间隔区约为800 bp，同源性非常高（> 99%）。从该间隔区推导得出两对可用于巢式PCR的属特异性引物对BRU - P5 - BRU - P8和BRU - P6 - BRU - P7，以及三种属特异性DNA探针BRU - ICG2、BRU - ICG3和BRU - ICG4。分别通过PCR和线性探针分析（LiPA），用这两对引物和三种探针检测18株布鲁氏菌菌株及56株来自其他相关分类群的菌株，以此检验引物组和探针的特异性和敏感性。基于对牛奶成分的酶处理以及随后的PCR和LiPA杂交，开发了一种直接检测1 ml生牛奶中布鲁氏菌属细菌的方法。单次PCR后，通过琼脂糖凝胶电泳和LiPA检测的灵敏度分别为2.8×10⁵CFU/ml和2.8×10⁴CFU/ml。两种方法的巢式PCR灵敏度均为2.8×10²CFU/ml。
