Identification of Mycobacterium Species Using Molecular Methods

Identification of Mycobacterium spp to the species level as per ' LPSN (List of Prokaryotic names with Standing in Nomenclature, author: J.P. Euzeby).' The testing is conducted using a combination of PCR and DNA sequence-based methodologies.

Solid or liquid media growth.

• For solid media growth, isolated colonies on plated media are preferred.

• For liquid culture, a minimum volume of 4 mL of actively growing culture is preferred.

• Mycobacterium tuberculosis complex and slowly growing Mycobacterium species must show visible growth and be no more than 4 weeks old.

• Rapidly growing Mycobacterium species must show visible growth and be no more than 10 days old.

• If there is inadequate growth, or there is contamination, submitted cultures will be rejected and a resubmission will be requested.

N/A

Ship all cultures at room temperature (DO NOT freeze) for overnight delivery, and prior to Wednesday each week to ensure receipt by Friday.

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.

None

Requisitions must be faxed prior to shipping isolates to NRCM - attention Joyce Wolfe, Program Manager, NRCM/NML at 204-789-2036. Requisition for the NRCM must be completed and signed off by the supervisor/designate of the submitting laboratory and include the source of specimen, patient gender, date of birth, clinical history, submitting laboratory identifier and submitter information. Also include isolate characteristics: microscopy, pigmentation, culture characteristics, growth rate/temperature, and other identification. Requested testing for urgent submissions should be accompanied with justification.

Cultures will also be rejected if appropriate documentation and justification is incomplete or missing.

Species identification will be determined through sequence analysis of the gyrB, 16S rRNA and/or hsp65 genes, as needed. The identification will be determined by comparison of sequence data to an in-house quality controlled databases.

Urgent submissions: 4 calendar days from the date of specimen receipt. Routine testing from culture: 10 calendar days from the date of specimen receipt.

1. Turenne CY, Tschetter L, Wolfe J, Kabani A. (2001) Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species. J Clin Microbiol. 39:3637-48.

2. Brunello F, Ligozzi M, Cristelli E, Bonora S, Tortoli E, and Fontana R. (2001) Identification of 54 mycobacterial species by PCR-restriction fragment length polymorphism analysis of the hsp65 gene. J. Clin. Microbiol. 39:2799-2806.