Random amplified polymorphic DNA (RAPD) analysis represents a special variation of PCR that can be regarded as gross whole-genome characterization. It consists in parallel amplification of an arbitrary set of genomic segments using short oligonucleotide primers of 8–12 nucleotides [4]. The amplification reaction involves one or more primers and is conducted at low-stringency conditions. The low annealing temperature allows primer binding to multiple sites that do not need to be completely complementary, so that the numbers and positions of binding sites are unique for each bacterial strain. Numerous amplicons in the range of 0.1–3 kbp can be produced and subsequently visualized using agarose gel electrophoresis.

RAPD has been used for discriminatory analysis of isolates of many microbial pathogens. For instance, investigating Mycoplasma bovis infections in cattle, the number of distinct strains present in a geographical region or a herd was determined based on the obtained banding patterns [5, 6]. This included identification of the strain at the source of the outbreak [7]. Likewise, strains of Mycobacterium (M.) avium were distinguished by their fingerprints using a panel of four or six primers [8, 9]. Differentiation among M. avium subsp. paratuberculosis strains using RAPD was also attempted [10], but proved more difficult.

The basic asset of the method is its versatility, which allows the use of a theoretically unlimited number of primers and thus offers unlimited capacity for revealing strain-to-strain differences [9]. It also means that the methodology can be adapted individually to any organism. While the method is easy to use, inexpensive, and rapid, its major drawback consists in limited reproducibility. Banding patterns tend to vary considerably from run to run and from laboratory to laboratory. Therefore, it was recommended to conduct RAPD analyses in triplicate [8].

Restriction fragment length polymorphism (RFLP) analysis is based on enzymatic cleavage of genomic DNA of an isolate. The fragments generated by specific restriction endonucleases are separated according to their size using gel electrophoresis. Mutations in the genome can lead to changes in the number of cleavage sites, whereas insertions and deletions can shift their positions and give rise to “fragment length polymorphisms.”

A well-known example of RFLP typing refers to Campylobacter jejuni, where the tandem flagellin genes flaA and flaB served as target for subtyping in molecular epidemiological studies [11]. The classical serovars of Chlamydia psittaci could be distinguished using PCR-RFLP of the ompA gene locus [12].

If an organism’s genome carries specific insertion sequences, Southern hybridization with a fluorescent-labeled complementary gene probe can be used to reveal banding patterns that are specific for individual strains. Thus, IS6110-RFLP was widely used for genotyping strains of the Mycobacterium tuberculosis complex (MTC), e.g., M. tuberculosis [13], M. bovis, and M. caprae [14]. The discriminatory capacity is particularly high for strains with six or more IS6110 copies [15]. In the case of M. avium subsp. paratuberculosis, RFLP based on IS900 and using at least two restriction enzymes can provide high resolution in typing [16, 17]. Ribotyping [18], which was used for years as a versatile typing method for bacterial families, genera, and species, also includes the use of rDNA probes, so that only those bands containing a portion of the ribosomal operon are visualized. The number of reactive bands on Southern blot reflects the multiplicity of rRNA operons in a microbial species. Numerous applications to taxonomic classification, epidemiological tracking, geographical distribution, population biology, and phylogeny were reported [19]. The general use of RFLP-based typing has been in decline for the last few years, not so much for being labor-intensive, time-consuming, and technically demanding, but mainly because more efficient assays have become available (see below).

Amplified fragment length polymorphism (AFLP) analysis is based on selective PCR amplification of restriction endonuclease-digested genomic DNA [20]. The cleavage reaction involving one, (typically) two, or more enzymes is followed by ligation of oligonucleotide adaptors on both termini of the fragments. Subsequent PCR often includes two amplification steps and uses primers complementary to the adaptor sequences to amplify a subset of the restriction fragments. Individual assays can be tailored by using selective bases in the adaptor sequences to keep the number and size of finally generated amplicons in a manageable range, e.g., 50–100 fragments of 50–500 bp. Visualization of the patterns is accomplished through acrylamide gel or capillary electrophoresis.

In a comparative study on Mycoplasma bovis isolates from cases of respiratory disease in calves from different regions of the United Kingdom, McAuliffe et al. [5] identified two genetically distinct clusters, whereupon the AFLP-PCR findings largely coincided with those of RAPD. Wagenaar et al. [21] used AFLP-PCR for genotyping of Campylobacter fetus strains and were able to differentiate the subspecies Campylobacter fetus subsp. venerealis. Hu et al. [22] identified phage type-specific markers by using this technique for Salmonella Typhimurium strains.

The advantages of AFLP-PCR include its high sensitivity and resolution for genome-wide detection of polymorphisms, as well as relatively good reproducibility. On the other hand, the number of amplified fragments has to be limited in order to keep the performance at high level. All in all, the procedure is relatively elaborate and requires purified and intact double-stranded DNA, as well as specialized equipment and software.

The acronym stands for macro-restriction fragment length polymorphism analysis using pulsed-field gel electrophoresis. The method utilizes specific restriction sites throughout the microbial genome for differentiation below the species level. Bacterial cultures are embedded in agarose blocks, lysed in situ, and digested with rare-cutter restriction endonucleases. The resulting macro-restriction fragments sized up to 10 Mb can only be separated using agarose gel electrophoresis with an alternating electric field [23]. The final fingerprint patterns typically consist of 10–20 fragments. For an update on recent developments in PFGE technology, the reader is referred to an exhaustive review [24].

Due to its high discriminatory power, PFGE analysis evolved as a gold standard for typing of many bacteria, such as Campylobacter spp. [25], Clostridium spp. [26, 27], and Salmonella [28]. There are publicly accessible databases having thousands of individual strain patterns of food-borne pathogens, such as Salmonella serovars, Listeria monocytogenes, and others (e.g., http://​www.​pulsenetinternat​ional.​org/​).

The main reason why the use of the procedure has been confined to a limited circle of specialized laboratories lies in its sophisticated and technically demanding work flow. For instance, the in situ digestion of genomic DNA in the agarose block may prove difficult for certain pathogens. Therefore, it remains a challenge to attain satisfactory interlaboratory reproducibility.