Gene Sequencing - 16S rRNA

16S rRNA gene PCR, sequencing, and phylogenetic analysis. Secondary gene target sequencing and phylogenetic analysis is used complementarily with 16S gene sequencing. The choice of specific secondary gene target is dependent on 16S gene sequencing results.

Pure cultures of isolate implicated in clinical illness or environmental isolates.

Slants or plates of any suitable media and swabs in transport media are all acceptable. Samples submitted for isolation and/or identification of strict anaerobes must be submitted under oxygen-free conditions. Pre-reduced anaerobically sterilized (PRAS)/Cary and Blair transport medium or PRAS Cooked Meat broth are acceptable.

Store and ship samples at room temperature.

Shipping of specimens shall be done by a TDG certified individual in accordance with TDG regulations. For additional information regarding classification of specimens for the purposes of shipping, consult either Part 2 Appendix 3 of the TDG Regulations or section 3.6.2 of the IATA Dangerous Goods Regulations as applicable.

Clinical illness with symptoms suggestive of bacterial infection.

Completed Special Bacteriology requisition form detailing all patient information and relevant clinical information. If possible, attach lab results that have already been done at local or provincial laboratories.

All patient and strain history must be included. Enteric bacteria, Mycobacteria, common nosocomial agents (MRSA, VRE), Neisseria, Eukaryotes, and Risk Group 3 bacteria are NOT accepted by Special Bacteriology. Please refer to the Guide to Services for test services offered by the NML.

16S gene and secondary gene target (if applicable) sequencing results are compared against publically-available databases (GenBank) and known type strains. In a case when subspecies exist, some traditional phenotypic testing may be done. The latter is solely at the discretion of the Special Bacteriology Laboratory.

12 calendar days for the 16S/secondary gene target final report. Report may be delayed if phenotypic testing needs to be done. In cases where staff or resources are limited, or for poor or slow growing organisms, final report may be delayed and status of request will be forwarded in a preliminary report. Final report is sent once all testing is complete.

1. Baldwin et al. 2005. Multilocus Sequence Typing Scheme That Provides Both Species and Strain Differentiation for the Burkholderia cepacia Complex. JCM 43(9): 4665-4673.
2. Conville PS, Zelazny AM, Witebsky FG. 2006. Analysis of secA1 gene sequences for identification of Nocardia species. J Clin Microbiol. 44(8):2760-6.
3. Khamis, A, Raoult, D, La Scola, B. 2004. rpoB Gene Sequencing for Identification of Corynebacterium Species. JCM 42: 3925-3931.
4. Kolbert CP, Rys PN, Hopkins M, Lynch DT, Germer JJ, O’Sullivan CE, et al. 16S ribosomal DNA sequence analysis for identification of bacteria in a clinical microbiology laboratory. In: Persing DH, Tenover FD, Versalovic J, Tang Y-W, Unger ER, Relman DA, White TJ, eds. Molecular microbiology: diagnostic principles and practice. Washington, DC: ASM Press 2004. p 361–77.
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6. Yamamoto, S, PJM Bouvet, and S Harayama. 1999. Phylogenetic structures of the genus Acinetobacter based on gyrB sequences: comparison with the grouping by DNA-DNA hybridization. International Journal of Systematic Bacteriology. 49: 87-95.