AFLP protocol comparison for microbial diversity fingerprinting
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Over the last decade, several methods based on genomic DNA have been developed for the identification and genotyping of prokaryotic and eukaryotic organisms. These genomic methods differ regarding taxonomic range, discriminatory power, reproducibility, and ease of interpretation and standardization. The amplified fragment length polymorphism (AFLP) technique is a very powerful DNA fingerprinting technique for DNA of any source or complexity, varying in both size and base composition. In addition, this method shows high discriminatory power and good reproducibility allowing it to be efficient in discriminating at both the species and strain levels. The development and application of AFLP have allowed significant progress in the study of biodiversity and taxonomy of microorganisms. In the last years, the Applied Biosystems AFLP Microbial Fingerprinting Kit, now out of production, was widely used in various studies to perform AFLP characterization of selected bacteria strains (described by Vos et al. (Nucleic Acids Res 23(21):4407–4414, 1995)). Its replacement gives the possibility for laboratories to continue the use of the previous AFLP data as a reference for bacteria genetic fingerprinting analysis in biodiversity studies. To overcome this issue a result comparison, by using an improved AFLP protocol and the AFLP commercial kit, was performed. In particular, previous results on different species (Listeria monocytogenes, Lactobacillus plantarum, and Streptococcus thermophilus) obtained with the commercial kit were compared with the improved AFLP procedure to validate the protocol. When compared with the AFLP Microbial Fingerprinting Kit, the improved protocol shows high reproducibility, resolution, and overall, is a faster method with lower costs.
KeywordsAFLP protocol Bacterial diversity Genome polymorphisms Phylogenetic analysis
Authors acknowledge the Department of Soil, Plant, and Food Science, University of Bari, Italy, for providing L. plantarum strains (POM1, POM31, POM43, POM40, POM8, C6, POM38). The authors are grateful to “Consorzio tutela Pecorino Toscano DOP” and “Consorzio tutela del formaggio Gorgonzola” for providing the samples.
Bertani contributed to the analysis, acquisition, and interpretation of data; drafting of the manuscript; and critical revision. Savo Sardaro contributed to the study conception and designs; the work, analysis, and interpretation of data; drafting of the manuscript; and critical revision. Lazzi and Neviani contributed to design the work and in the critical revision.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with animals performed by any of the authors.
- Di Cagno R, Minervini G, Sgarbi E, Lazzi C, Bernini V, Neviani E, Gobbetti M (2010) Comparison of phenotypic (biolog system) and genotypic (random amplified polymorphic DNA-polymerase chain reaction, RAPD-PCR, and amplified fragment length polymorphism, AFLP) methods for typing Lactobacillus plantarum isolates from raw vegetables and fruits. Int J Food Microbiol 143(3):246–253CrossRefGoogle Scholar
- Hamza AA, Robene-Soustrade I, Jouen E, Lefeuvre P, Chiroleu F, Fisher-Le Saux M, Gagnevin L, Pruvost O (2012) MultiLocus sequence analysis and amplified fragment length polymorphism-based characterization of xanthomonads associated with bacterial spot of tomato and pepper and their relatedness to Xanthomonas species. Syst Appl Microbiol 35(3):183–190CrossRefGoogle Scholar
- Heir E, Lindstedt B-A, Vardund T, Wasteson Y, Kapperud G (2000) Genomic fingerprinting of shigatoxin-producing Escherichia coli (STEC) strains: comparison of pulsed-field gel electrophoresis (PFGE) and fluorescent amplified-fragment-length polymorphism (FAFLP). Epidemiol Infect 125:537–548CrossRefGoogle Scholar
- Savelkoul PH, Aarts HJ, De Haas J, Dijkshoorn L, Duim B, Otsen M, Rademaker JL, Schouls L, Lenstra JA (1999) Amplified-fragment length polymorphism analysis: the state of an art. J Clin Microbiol 37(10):3083–3091Google Scholar
- Zabeau M, Vos P (1993) Selective restriction fragment amplification: a general method for DNA fingerprinting. European Patent Office. Publication 0 534 858 AlGoogle Scholar