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Chapter 30 : Identification of Bacteria by DNA Target Sequencing in a Clinical Microbiology Laboratory
Category: Clinical Microbiology; Bacterial Pathogenesis
Nucleic acid sequencing of various bacterial genes and other DNA targets has been used for determining the phylogeny of bacteria and for their identification. A brief overview of nucleic acid sequencing is shown in this chapter. DNA targets have conserved regions flanking variable regions that can be used to differentiate closely related bacterial species. The routine use of sequencing can greatly enhance the ability of the clinical microbiology laboratory to identify bacteria on many levels. Of consideration in the routine use of DNA target sequencing is the need for technical expertise and its cost. The chapter addresses preparation of DNA from pure culture. Certain conventional methods such as latex agglutination assays are quicker, simpler, and less expensive than DNA target sequencing for the identification of beta-hemolytic streptococci. Basic conventional methods perform well in identifying common isolates, such as Bacteroides fragilis group, Peptostreptococcus spp., and most Clostridium spp. DNA target sequencing can provide more accurate identifications, especially since databases from conventional methods often are not current and do not reflect the tremendous genetic diversity within anaerobic taxa. For agents of bioterrorism, i.e., Bacillus anthracis, Brucella spp., Clostridium botulinum, Francisella tularensis, and Yersinia pestis, 16S rRNA sequencing has varying utility. Molecular studies have enhanced our knowledge about the taxonomical diversity among bacteria and allowed better definition of the epidemiology of bacterial infections.
Dye-terminator cycle sequencing of amplified 16S rRNA gene. Purified PCR amplicon, sequencing primer, and limited concentrations of dideoxynucleotide triphosphates (ddNTPs) into which four different fluorescent dyes have been incorporated are mixed with unlabeled deoxynucleotides (dNTPs). Synthesis terminates whenever a ddNTP instead of a dNTP is incorporated into a new strand. Strands of various lengths are synthesized and labeled as the terminal ddNTP is incorporated into the strand. Accumulated fragments are separated according to size by electrophoresis. During electrophoresis, labeled products are visualized by fluorescence, with each of the four fluorescent dyes indicating which of the terminal ddNTPs have been incorporated. Combining the terminal ddNTP information with the fragment size allows the determination of sequence information. Reprinted from reference 58 with permission from the publisher.
Schematic for 16S rRNA located on the small ribosomal subunit (30S). Arrows indicate the conserved regions that serve as primer targets for PCR amplification and DNA sequencing of bacteria.
Phylogenetic representation of an unusual clinical isolate, such as this Wautersia sp., can provide useful information to the clinician if included with the patient results.
Microorganisms with indistinguishable 16S rRNA gene sequences and suggested supplemental phenotypic tests
Number of copies of 16S rRNA gene in bacterial pathogens derived from sequence data from bacterial genomes in GenBank and the Ribosomal RNA Operon Copy Number Database a
Frequently used primer sequences for gene sequence-based identification of bacteria
Comparison of features of various nucleotide sequence databases a