Identification and phylogenetic analysis of Lactobacillus using multiplex RAPD-PCR

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Accurate identification and genetic relatedness assessment of Lactobacillus species are crucial for various applications.
• Traditional methods like carbohydrate fermentation can be insufficient for differentiating closely related strains.

Purpose of the Study:

• To evaluate the efficacy of multiplex Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) for characterizing Lactobacillus isolates.
• To compare the discriminatory power of multiplex RAPD-PCR with carbohydrate fermentation profiles for assessing genetic relatedness.

Main Methods:

• Multiplex RAPD-PCR was employed using a combination of two 10-mer oligonucleotides in a single PCR reaction.
• DNA profiles were generated for various Lactobacillus strains, including gastrointestinal isolates and a stomach isolate (ML004).
• A dendrogram was constructed based on RAPD profiles to visualize genetic relatedness, which was then compared to carbohydrate fermentation data.

Main Results:

• Multiplex RAPD-PCR successfully generated unique DNA profiles, enabling the discrimination of all tested Lactobacillus strains.
• The generated RAPD profiles provided sufficient data to construct a dendrogram, clearly clustering gastrointestinal isolates together.
• Genetic relatedness inferred from the dendrogram showed that isolate ML004 (stomach) is closely related to Lactobacillus fermentum, a finding not easily achievable with carbohydrate fermentation.

Conclusions:

• Multiplex RAPD-PCR is a valuable and effective tool for the characterization and inference of genetic relatedness among Lactobacillus isolates.
• This molecular technique offers higher resolution compared to carbohydrate fermentation for differentiating and grouping Lactobacillus strains.