应用于临床实验室的细菌16S rRNA基因测序鉴定方法的优缺点

David

Journal of Clinical Microbiology, September 2007, p. 2761-2764, Vol. 45, No. 9

MINIREVIEW


16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls
应用于临床实验室的细菌16S rRNA基因测序鉴定方法的优缺点
J. Michael Janda* and Sharon L. Abbott
Microbial Diseases Laboratory, Division of Communicable Disease Control, California Department of Public Health, Richmond, California 94804

The use of 16S rRNA gene sequences to study bacterial phylogeny and taxonomy has been by far the most common housekeeping genetic marker used for a number of reasons. These reasons include (i) its presence in almost all bacteria, often existing as a multigene family, or operons; (ii) the function of the 16S rRNA gene over time has not changed, suggesting that random sequence changes are a more accurate measure of time (evolution); and (iii) the 16S rRNA gene (1,500 bp) is large enough for informatics purposes (12). In 1980 in the Approved Lists, 1,791 valid names were recognized at the rank of species. Today, this number has ballooned to 8,168 species, a 456% increase (http://www.bacterio.cict.fr/number.html#total). The explosion in the number of recognized taxa is directly attributable to the ease in performance of 16S rRNA gene sequencing studies as opposed to the more cumbersome manipulations involving DNA-DNA hybridization investigations. DNA-DNA hybridization is unequivocally the "gold standard" for proposed new species and for the definitive assignment of a strain with ambiguous properties to the correct taxonomic unit. Based upon DNA-DNA reassociation kinetics, the genetic definition of a species is quantifiable, i.e., (i) ca. 70% DNA-DNA relatedness and (ii) 5°C or less Tm for the stability of heteroduplex molecules. DNA hybridization assays are not without their shortcomings, however, being time-consuming, labor-intensive, and expensive to perform. Today, fewer and fewer laboratories worldwide perform such assays, and many studies describing new species are solely based upon small subunit (SSU) sequences or other polyphasic data.

In the early 1990s the availability DNA sequencers in terms of cost, methodologies, and technology improved dramatically, such that many centers can now afford such instrumentation. In 1994, Stackebrandt and Goebel (15) summarized the emergence of SSU sequence technology and its potential usefulness in the definition of a species. Although it has been demonstrated that 16S rRNA gene sequence data on an individual strain with a nearest neighbor exhibiting a similarity score of <97% represents a new species, the meaning of similarity scores of >97% is not as clear (13). This latter value can represent a new species or, alternatively, indicate clustering within a previously defined taxon. DNA-DNA hybridization studies have traditionally been required to provide definitive answers for such questions. Whereas 16S rRNA gene sequence data can be used for a multiplicity of purposes, unlike DNA hybridization (>70% reassociation) there are no defined "threshold values" (e.g., 98.5% similarity) above which there is universal agreement of what constitutes definitive and conclusive identification to the rank of species.