Goh S H, Potter S, Wood J O, Hemmingsen S M, Reynolds R P, Chow A W
Department of Medicine, University of British Columbia, Vancouver Hospital Health Sciences Centre, Canada.
J Clin Microbiol. 1996 Apr;34(4):818-23. doi: 10.1128/jcm.34.4.818-823.1996.
A set of universal degenerate primers which amplified, by PCR, a 600-bp oligomer encoding a portion of the 60-kDa heat shock protein (HSP60) of both Staphylococcus aureus and Staphylococcus epidermidis were developed. However, when used as a DNA probe, the 600-bp PCR product generated from S. epidermidis failed to cross-hybridize under high-stringency conditions with the genomic DNA of S. aureus and vice versa. To investigate whether species-specific sequences might exist within the highly conserved HSP60 genes among different staphylococci, digoxigenin-labelled HSP60 probes generated by the degenerate HSP60 primers were prepared from the six most commonly isolated Staphylococcus species (S. aureus 8325-4, S. epidermidis 9759, S. haemolyticus ATCC 29970, S. schleiferi ATCC 43808, S. saprophyticus KL122, and S. lugdunensis CRSN 850412). These probes were used for dot blot hybridization with genomic DNA of 58 reference and clinical isolates of Staphylococcus and non-Staphylococcus species. These six Staphylococcus species HSP60 probes correctly identified the entire set of staphylococcal isolates. The species specificity of these HSP60 probes was further demonstrated by dot blot hybridization with PCR-amplified DNA from mixed cultures of different Staphylococcus species and by the partial DNA sequences of these probes. In addition, sequence homology searches of the NCBI BLAST databases with these partial HSP60 DNA sequences yielded the highest matching scores for both S. epidermidis and S. aureus with the corresponding species-specified probes. Finally, the HSP60 degenerate primers were shown to amplify an anticipated 600-bp PCR product from all 29 Staphylococcus species and from all but 2 of 30 other microbial species, including various gram-positive and gram-negative bacteria, mycobacteria, and fungi. These preliminary data suggest the presence of species-specific sequence variation within the highly conserved HSP60 genes of staphylococci. Further work is required to determine whether these degenerate HSP60 primers may be exploited for species-specific microbic identification and phylogenetic investigation of staphylococci and perhaps other microorganisms in general.
开发了一组通用简并引物，通过聚合酶链反应（PCR）扩增出一段600碱基对的寡聚物，该寡聚物编码金黄色葡萄球菌和表皮葡萄球菌60千道尔顿热休克蛋白（HSP60）的一部分。然而，当用作DNA探针时，表皮葡萄球菌产生的600碱基对PCR产物在高严格条件下未能与金黄色葡萄球菌的基因组DNA交叉杂交，反之亦然。为了研究不同葡萄球菌中高度保守的HSP60基因内是否可能存在物种特异性序列，从六种最常见的葡萄球菌（金黄色葡萄球菌8325 - 4、表皮葡萄球菌9759、溶血葡萄球菌ATCC 29970、施氏葡萄球菌ATCC 43808、腐生葡萄球菌KL122和路邓葡萄球菌CRSN 850412）制备了由简并HSP60引物产生的地高辛标记的HSP60探针。这些探针用于与58株葡萄球菌和非葡萄球菌参考菌株及临床分离株的基因组DNA进行斑点杂交。这六种葡萄球菌的HSP60探针正确鉴定了所有葡萄球菌分离株。通过与不同葡萄球菌混合培养物的PCR扩增DNA进行斑点杂交以及这些探针的部分DNA序列，进一步证明了这些HSP60探针的物种特异性。此外，使用这些部分HSP60 DNA序列在NCBI BLAST数据库中进行序列同源性搜索，表皮葡萄球菌和金黄色葡萄球菌与相应物种特异性探针的匹配分数最高。最后，HSP60简并引物显示能从所有29种葡萄球菌以及30种其他微生物中的除2种以外的所有微生物中扩增出预期的600碱基对PCR产物，这些微生物包括各种革兰氏阳性和革兰氏阴性细菌、分枝杆菌和真菌。这些初步数据表明葡萄球菌高度保守的HSP60基因内存在物种特异性序列变异。需要进一步开展工作来确定这些简并HSP60引物是否可用于葡萄球菌以及可能其他微生物的物种特异性微生物鉴定和系统发育研究。
