The Use of AFLP for DNA Fingerprinting

Over the last decade, for the identification and genotyping of prokaryotic and eukaryotic organisms, several methods at the DNA level have been developed. These genomic methods differ as regard taxonomic range, discriminatory power, reproducibility, and ease of interpretation and standardization. The Amplified Fragment Length Polymorphisms (AFLP) technique is a very powerful DNA fingerprinting technique for DNAs of any source or complexity, varying in both size and base composition. In addition, this method shows high discriminatory power and good reproducibility showing to be efficient for discriminating at the species but also at the strain level. The development and application of AFLP have allowed to significant progress in the study of biodiversity and taxonomy of microorganisms. In the last years, the Applied Biosystems AFLP Microbial Fingerprint Kit protocol was widely used in various studies to perform AFLP characterization of selected bacteria strains, previously described by Vos et al. (1995), although including several modifications. This study aims to propose an alternative protocol to replace the commercial kit out of production, giving so the possibility to use the method for bacteria genetic fingerprinting analysis in biodiversity study. In particular previous results on different species (Listeria monocytogenes, Lactobacillus plantarum and Streptococcus thermophilus) obtained with the commercial kit were compared with new AFLP procedure to validate the protocol. In comparison with the AFLP Microbial Fingerprint Kit, the new designed protocol shows high reproducibility, resolution and it is a faster method with lower costs.

Key words: AFLP protocol,Bacterial diversity,GenomePolymorphisms, Phylogenetic analysis, Micro-biology

Introduction

In the last decades, several PCR-based fingerprinting methods have been developed for bacteria genomic fingerprinting and genome screening purposes. It is possible to differentiate high-throughput approaches based on DNA or RNA analyses based on several next-generation sequencing techniques (Ercolini et al. 2015; De Filippis et al. 2017; Garofalo et al. 2017) and the low-throughput methodologies that are more largely used. Among the last, can be considered the randomly amplified polymorphic DNA (RAPD) (Cocconcelli et al. 1995; Franklin et al. 1999; Rossetti and Giraffa, 2005; Perin et al. 2017), the primers based on repetitive elements in the genome (rep-PCR) (Versalovic et al. 1991; De Bruijn, 1992; Anderson et., 2010), the amplified ribosomal DNA restriction analysis (ARDRA) (Vaneechoute et al. 1992; Gulitz et al. 2013), the automated ribosomal intergenic spacer analysis (ARISA) (Kovacs et al. 2010) and length heterogeneity-PCR (LH-PCR) (Lazzi et al. 2004, Gatti et al. 2008, Savo Sardaro et al. 2018). Besides these, Amplified fragment length polymorphism (AFLP) (Zabeau and Vos, 1993; Vos et al. 1995; Janssen et al. 1996) has been largely used for genomic fingerprinting of DNA from a variety of sources. As widely reported in literature, AFLP is a valuable technique for the classification of bacteria at the species and strain level with high discriminatory power and good reproducibility (Janssen et al. 1996; Blears et al. 1998; Savelkoul et al. 1999; Jarraud et al. 2002). The AFLP fingerprinting provides several advantages over other techniques (Curtin et al. 2007). First of all, prior knowledge of a microorganism’s genome sequence is not necessary. Moreover, AFLP alleles can be fluorescently labelled, allowing a parallel characterization of several samples in automatic genome analysers. Once the technique is fine-tuned, it is possible to obtain accurate information fast enough to allow for an efficient identification and differentiation of species and strains. The development and application of AFLP as a fingerprinting method has led to significant progress in the study of the genetic diversity and taxonomy of bacteria (Heir et al. 2000; Giraffa et al. 2001; Cocolin and Ercolini, 2008; Cappello et al. 2008; Lazzi et al. 2009; Di Cagno et al. 2010; Lévesque et al. 2012; Nabhan et al. 2012; Hamza et al. 2012; Bernini et al. 2013; Jérôme et al. 2016). Most of the bacteria strains characterization, in the last years, was developed following the Applied Biosystems AFLP Microbial Fingerprint Kit protocol, according to the manufacturer’s instructions. In this paper, is reported an improvement of the AFLP protocol taking into consideration the increasing of the alleles amplification efficiency and resolution, time-saving and cost effectiveness, comparing the advanced protocol with the results obtained using the commercial AFLP microbial kit.

Materials and methods

Bacteria and growth conditions

Twenty-one strains isolated from different food matrices were used in this study (Table 1). They include 7 Streptococcus thermophilus, 7 Listeria monocytogenes and 7 Lactobacillus plantarum strains. Bacterial strains were maintained as frozen stocks (−80 °C) in M17 (S. thermophilus), TSB broth (L. monocytogenes) and MRS (L. plantarum) (Oxoid, Milan, Italy) supplemented with 15% glycerol (w/v). Before use, the cultures were propagated twice with a 3% (v/v) inoculum into the appropriate media and incubated at 42°C (S.thermophilus), 37°C (L. monocytogenes) and 30°C (L. plantarum) for 24 h in optimal growth conditions. All S. thermophilus and L. monocytogenes strains belong to the collection of Food and Drug Department, University of Parma, Italy; seven L. plantarum strains (POM1, POM31, POM43, POM40, POM8, C6, POM38) were kindly given by the Department of Soil, Plant and Food Science, University of Bari, Italy.

AFLP analysis

Preparation of primary template for AFLP analysis

The AFLP procedure was performed according to the method of Vos et al. (1995) with the modifications described below. Restriction-Ligation reactions were performed in a final volume of 50 µl containing 1×T4 DNA ligase buffer with 1 mM ATP, 250 ng/µl BSA, 10 mM ATP, 20 U/µl of EcoRI, 10 U/µl of MseI and 500 ng of genomic DNA. Two different adapters (sequences shown in Table 2), one for the EcoRI sticky ends and one for the MseI sticky ends, were ligated to the DNA by adding to the reaction of a mix containing 5 pmol/µL of EcoRI adaptor, 50 pmol/µl of MseI adaptor, and 200 U/µl of T4 DNA ligase (New England Biolabs). The reaction was incubated for 4h at 37°C. Two replication for each sample were performed.

The digested-ligated DNA product to be used as templates for the first amplification reaction was diluted 10 fold with RNAse and DNAse free water and stored at -20 °C.

Pre-amplification

The “non-selective” primers EcoRI-0 and MseI-0 (Table 2) were used for pre-amplification of digested-ligated DNA. Each pre-amplification contained 5 μl of digested-ligated DNA previously described, 1.5 μl of unlabelled MseI-0 primer (10 μM) and 1.5 μl of labelled EcoRI-0 primer (10 μM), 25 μl of GoTaq® Colorless Master Mix (PROMEGA, Madison, Wisconsin, Stati Uniti).

The reaction was subjected to the following PCR conditions: 3 min at 94 °C, 14 cycles (45 s at 94 °C, 30 s at 65 °C and in each cycle the annealing temperature decrease 1°C, 1 min at 72°C), 19 cycles (45 s at 94°C, 30 s at 56°C and 1 min at 72°C), 5 min extension at 72°C and a final step for 15 min at 30 °C. All amplifications were performed in a GeneAmp® PCR System 2700 (Applied Biosystem). Subsequently, the pre-amplification product was diluted 10 fold with RNAse and DNAse free water and stored at -20 °C.

Selective-amplification

Different primer combinations were used, on the basis of different species analysed: EcoRI-A/MseI-C for L. monocytogenes and EcoRI-A/MseI-A for S. thermophilus and L. plantarum (Table 2). Each selective-amplification contained 5 μl of the diluted pre-amplification product described previously, 1.5 μl of unlabelled MseI-A primer (10 μM) and 1.5 μl of labelled EcoRI-A primer (10 μM), 25 μl of GoTaq® Colorless Master Mix (PROMEGA).

The thermocycler program consisted in 2 min at 72 °C, 33 cycles (30 s at 94 °C, 1 min at 56 °C, 1 min at 72 °C), 2 min extension at 72°C and a final step for 30 min at 60 °C. All amplifications were performed in a GeneAmp® PCR System 2700 (Applied Biosystem). Ten microliters of each selective amplification product were separated by electrophoresis on a 1.2% agarose gel at 90 V/cm for 20 min to check the amplifications.

Fragments analysis

Eight microliters of each amplified products from selective amplification were added to 1.5 μl of GeneScan-500 [LIZ] size standard (Applied Biosystem-Pe Corporation) and 27 μl of deionized formamide. The mixture was heated 5 min at 95°C and cooled on ice. Samples were loaded and run on the ABI Prism 310 (Applied Biosystem-Pe Corporation,Waltham, Massachusetts, USA) and analysed using GeneMapper Analysis Software (Applied Biosystem-Pe Corporation). The data for each run were saved as an individual GeneScan file and displayed as an electropherogram. A threshold, used in scoring to consider only sharp and easily distinguishable peaks, of 50 RFU (Relative Fluorescent Unit) was considered for results obtained by new protocol, while a threshold of 80 RFU for results obtained with AFLP Microbial Fingerprinting Kit; all the signals under this value were treated as background and not scored. Peaks representing AFLP fragments from 50 to 500 bp were reported as binary format with “1” for the presence of a band and “0” for its absence.

Results and Discussion

This study focuses on the develop an advanced AFLP procedure for the detection of polymorphisms in bacterial genomes (Zabeau & Vos, 1993; Vos et al.1995; Janssen et al. 1996). The new advanced procedure was mainly compared with AFLP Microbial Fingerprinting Kit, considering  its wide employment, in the last years, for bacteria AFLP fingerprinting analysis. The main aim has been to develop an AFLP protocol, to replace the AFLP Microbial Fingerprinting Kit, able to improve the profile quality of the assay, increasing sensitivity and precision and decreasing scoring time and errors.

The first modification in the protocol, in comparison to AFLP Microbial Fingerprinting Kit, is related to performing a combined digestion and adaptor ligation at the same time without affecting the number of final bands and the final results. The experiment was performed on the 21 strains showed in table 1(Supplementary material) and an example of the result is presented in Fig. 1 where the comparison between the advanced protocol and the commercial kit on the strain Lm44 showed the same profiles. This modification gives the opportunity to save time and to reduce the laboratory costs for analysis and this was also considered positively by Curtin et al. (2007). The second condition that has been also evaluated for modification, is the dilution of the digested-ligated DNA fragments to be used as templates in the first amplification reaction. The dilution in AFLP Microbial Fingerprinting Kit procedure is 20 fold while in the optimized AFLP protocol this dilution of digested-ligated DNA fragments has a negative influence in the electropherograms resolution, whereas the optimal dilution of digested-ligated DNA fragments is obtained with 10 fold. Figure 2 shows the electropherograms obtained with the two dilutions using the strains S. thermophilus 100 and 4042.

The third parameter that was consider is the one related with the dilution of the pre-selective PCR products used as templates for the subsequent selective PCR. Two different conditions were evaluated one without any dilution in compare with samples diluted 10 fold. The amplification results analysed by capillary electrophoresis were very similar one each other (data not shown) so for this parameter the condition reported by AFLP Microbial Fingerprinting Kit and other authors (Vos et al. 1995; Janssen et al. 1996) of 10 fold dilution, was maintained.

Finally, the PCR conditions have been modified with the touchdown PCR applied in the pre-amplification reaction and not in the selective one as in the kit. In addition the number of PCR cycles in the selective amplification has been taken in consideration with the aim to increase the peak intensity without introduce high level of Taq polymerase errors, that can give also differences in peak base pair size. The number of selective PCR cycles has been increased to 33 comparing with Vos et al. (1995), Janssen et al. (1996) and the commercial kit where this parameter was 24 and 30 respectively. The modification introduced allows us to have electropherograms with peaks higher in the intensity of fluorescence and more defined for the next step of the data elaboration (Fig. 3). In addition, the new optimized protocol provides an improvement in the signal-to-background ratio in the electropherograms and increase the intensity of the peaks profiles obtained (Fig 4 and Supplementary material). Also, the possibility to maintain and compare previous data obtained with the AFLP microbial kit is showed in Fig.4 where two strains previously analysed, by Lazzi et al. (2009) using the commercial kit, have the same peaks profiles with the advanced AFLP protocol and it is also shown in supplementary material on the other 19 strains. This opens the possibility to all laboratory to still continue their phylogenetic study using data previously obtained with the commercial kit.

Conclusion

AFLP is an excellent technique to differentiate strains or very closely related species and as a good phylogenetic tool. In the last years, the use of several restriction enzymes and many fluorescence molecules at the same time has given the opportunities to achieve a very extensive screening of the bacteria genomes. The modification of the digested-ligated step reduces drastically the time needed for the sample’s analysis. Moreover, the different condition of dilution of digested-ligated DNA fragments and the possibility to increase the number of PCR cycles allow to obtain comparable and better results in terms of distinctiveness and intensity of the band’s peaks in comparison with the commercial AFLP Microbial kit. In addition, considering that this kit is not available anymore and the resulted electropherograms are the same with both protocols, thanks to this method we can compare AFLP profiles with previous database without repeating the analysis of all the samples.

Overall the modified protocol gives the opportunities to reduce the time-consuming and labour-intensive, and costs effective given the possibilities to continue to use the AFLP technique as an excellent tool to analyse a large number of samples.

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Tables

Table 1 Strains used in this study.

Table 2 Primers used for AFLP analysis.

Figures captions

Figure 1. Electropherograms of L. monocytogenes strain LM44 AFLP profiles. Comparison of AFLP profiles obtained with combined and not-combined digestion-ligation procedure. A) AFLP profile strain LM44 obtained with not-combined digestion-ligation procedure using AFLP Microbial Fingerprinting kit; B) AFLP profile strain LM44 performed with combined digestion-ligation procedure in the advanced AFLP protocol.

Figure 2. Electropherograms of S. thermophilus strains 100 and 4042 AFLP profiles obtained using 20 fold and 10 fold dilution of digested-ligated DNA product. A) AFLP profile of strain 100 related to 20 fold dilution of digested-ligated DNA product; B) AFLP profile of strain 100 related to 10 fold dilution of digested-ligated DNA product; C) AFLP profile of strain 4042 related to 20 fold dilution of digested-ligated DNA product; D) AFLP profile of strain 4042 related to 10 fold dilution of digested-ligated DNA product.

Figure 3. Electropherograms of S. thermophilus strains 201 and 100 AFLP profiles of the PCR product obtained using 33 and 30 PCR cycles in selective PCR. A) AFLP profile of strain 201 related to 30 cycles in selective PCR; B) AFLP profile of strain 201 related to 33 cycles; C) AFLP profile of strain 100 related to 30 cycles in selective PCR; D) AFLP profile of strain 100 related to 33 cycles in selective PCR.

Figure 4. Comparisons of the AFLP profiles of S. thermophilus strains 4027 and 4028 obtained with the AFLP microbial Kit and the advanced AFLP protocol. A) AFLP profile of strain 4027 obtained with the AFLP microbial Kit; B) AFLP profile of strain 4027 obtained with the advanced AFLP protocol; C) AFLP profile of strain 4028 obtained with the AFLP microbial Kit; D) AFLP profile of strain 4028 obtained with the advanced AFLP protocol.