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DNA fingerprinting of mycobacteria using automated repetitive sequenced-based PCR

Abstract number: 10.1111/j.1198-743X.2004.902_o187.x

Lising M.

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Objective:

DNA fingerprinting of mycobacterial pathogens, including Mycobacterium tuberculosis (Mtb), Mycobacterium avium complex (MAC), and nontuberculous mycobacteria (NTM), has numerous applications in clinical microbiology and molecular epidemiology. Current genotypic methods used to type Mtb, MAC and NTM strains include RFLP, PFGE, Spoligotyping, and MIRU-based typing. Additionally, identification of mycobacteria other than tuberculosis (MOTT) is challenging and may include sequencing or traditional biochemical testing. These methods are either time consuming, laborious, expensive, or highly restricted with regard to applicability. This study reports the use of a simple, rapid and cost-effective rep-PCR technology for typing isolates of most mycobacterial species using the DiversiLab System.

Method:

Fingerprints from 25 different ATCC Mycobacterium species were stored in a library. Twenty-five clinical isolates were gathered from two geographical regions and were grown on Lowenstein–Jensen media. DNA from each culture was extracted using the UltraCleanTM Microbial DNA Isolation Kit. Seventy-five nanograms of each sample DNA was used to generate rep-PCR-based fingerprints using the DiversiLab Mycobacterium Kit, microfluidic chips, and the Agilent 2100 analyser. Sample analyses and data archiving were carried out using the DiversiLab System.

Result:

Dendrogram and gel-like images of rep-PCR profiles for all 25 different ATCC Mycobacterium species and all MOTT clinical isolates were obtained using DiversiLab System. The rep-PCR clustering identified MOTT strains and showed excellent concordance with similar ATCC species and biochemical characterization. Similarly, different isolates of rapidly growing mycobacteria and slow-growing NTM were fingerprinted using the DiversiLab Mycobacterium Kit.

Conclusion:

Rep-PCR has distinctive advantages for mycobacterial typing such as ease of use, requirement of relatively small amounts of DNA, the potential for typing strains not typeable by RFLP due to low IS6110 copy number, and the ability to type other mycobacterial species. Based on this pilot study, the DiversiLab System shows considerable promise as a tool for identification and typing of MOTT.

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Session Details

Date: 01/08/2007
Time: 00:00-00:00
Session name: XXIst ISTH Congress
Subject:
Location: Oxford, UK
Presentation type:
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