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Development of a low-cost and robust assay for the screening of multiple genetic loci in Mycobacterium tuberculosis

Abstract number: 1732_128

Bergval I.L., Anthony R.M., Dalla Costa E.R., Schuitema A.R.J., Oskam L., Klatser P.R.

Objective: The rapid characterisation of (drug-resistant) Mycobacterium tuberculosis (MTB) would be useful for the research and treatment of tuberculosis (TB). Many important genetic markers of MTB have been identified, notably for drug resistance, but also genotype, bacterial lineage and adaptive potential. A single assay allowing identification of these markers would aid control efforts. Multiplex PCR is unsuitable for simultaneous amplification of several genetic loci in one reaction. With Multiplex Ligation-dependent Probe Amplification (MLPA) only one primer-pair is used for the amplification of multiple genetic targets.

We explored the utility of MLPA as a screening tool to rapidly characterise MTB-strains

Method: MLPA can identify multiple single nucleotide polymorphisms (SNPs) by amplification of sequence-specific MLPA-probes, rather than target DNA. A sensitive ligation-step ensures specificity of the assay. We designed MLPA-probes specific for a range of discriminatory SNPs in MTB. In addition, we selected three sequences of conserved regions to use as internal controls and for quantitation. The size of MLPA-products corresponds to the specific SNP they targeted.

Amplified MLPA-products were identified on an agarose-gel or by fragment analysis, using capillary electrophoresis.

Results: Probes were initially validated on DNA from reference strains. All probes were then combined in a single mixture and used to characterise laboratory strains and a panel of clinical isolates from Brazil, Peru and the Netherlands. To determine the predictive value of MTB-specific MLPA, we screened DNA of MTB-strains with unknown genotypes. Results were confirmed by sequencing.

There was a very high correlation between sequencing results and results obtained via MLPA. In addition, both the positive and the negative predictive value were high. MLPA-products were clearly visible on an agarose gel.

Conclusion: We have shown that with MTB-specific MLPA it is possible to identify drug resistance associated mutations and MTB lineage specific SNPs in a single assay. Depending on the application, probes can be added to or removed from the probe-mix, making it a flexible, multi-purpose method. MLPA products can be easily identified on an agarose-gel, making it especially suitable for screening of TB in the less-developed world.

Based on our results we feel that in addition to using MLPA on DNA extracted from culture, performing MLPA directly on DNA from sputum samples is also feasible.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Subject:
Location: ICC, Munich, Germany
Presentation type:
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