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Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp.
Alongkorn Amonsin1, Ling Ling Li, Qing Zhang
1Department of Microbiology and Biomedical Genomics Center, University of Minnesota, St. Paul, Minnesota 55108, USA.
Journal of Clinical Microbiology
|April 9, 2004
Summary
A new multilocus short sequence repeat (MLSSR) sequencing method effectively genotypes Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains. This high-resolution subtyping aids molecular epidemiology and population genetic studies of M. paratuberculosis.
Area of Science:
- Microbiology
- Genetics
- Epidemiology
Background:
- Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) causes Johne's disease in livestock.
- Accurate strain differentiation is crucial for understanding M. paratuberculosis transmission and epidemiology.
- Existing genotyping methods like MPIL and AFLP have limitations in resolution or reproducibility.
Purpose of the Study:
- To develop and validate a novel multilocus short sequence repeat (MLSSR) sequencing approach for high-resolution genotyping of M. paratuberculosis.
- To assess the discriminatory power of MLSSR for differentiating M. paratuberculosis isolates from various sources.
- To evaluate MLSSR as a tool for molecular epidemiologic and population genetic analyses.
Main Methods:
- Identification and selection of 11 polymorphic short sequence repeat (SSR) loci from the M. paratuberculosis genome.
- Comparative nucleotide sequencing of these 11 SSR loci across multiple M. paratuberculosis isolates.
- Genotyping of 33 M. paratuberculosis isolates using the selected MLSSR loci.
- Comparison of MLSSR results with existing multiplex PCR for IS900 loci (MPIL) and amplified fragment length polymorphism (AFLP) typing.
Main Results:
- The MLSSR approach identified 11 polymorphic SSR loci with an average of 3.2 alleles per locus.
- MLSSR differentiated 33 M. paratuberculosis isolates into 20 distinct types, achieving an index of discrimination of 0.96.
- MLSSR successfully distinguished between sheep and cattle isolates and differentiated major MPIL and AFLP genotypes.
- The method demonstrated high reproducibility and ease of use.
Conclusions:
- MLSSR sequencing provides a facile, reproducible, and high-resolution method for M. paratuberculosis strain subtyping.
- MLSSR is a valuable tool for molecular epidemiologic investigations and population genetic studies of M. paratuberculosis.
- This approach enhances our ability to track the spread and understand the population structure of M. paratuberculosis.