PCR employs thermostable polymerases to enable amplification by continuous thermocycling and is currently the most commonly used method for amplification of genetic material [3]. The highly charged phosphodiester backbone of DNA makes the PCR product amenable to high-resolution visualization on agarose gel electrophoresis utilizing DNA-binding fluorescent dyes such as ethidium bromide. Electrophoresis both provides a means for detection by band visualization and enables at least a tentative verification of specificity by estimation of the amplicon length.

Shortly after the introduction of PCR, attempts were made to enhance sensitivity of detection of target nucleic acid sequences by running a second PCR assay targeting the internal region of the amplicon resulting from the first reaction, so-called nested PCR [4, 5]. The greater sensitivity has been attributed to both a dilution effect of any inhibitory compounds present in the sample, since only a minor fraction of the first reaction volume is used in the second reaction, and the fact that the primer-driven reaction is run twice, using four specific primers, rather than two. An intermediate situation is obtained if one of the primers from the first reaction is retained in the second, which yields a semi-nested PCR format.

The drawback of using PCR, and in particular the nested PCR formats, is that conserved regions must exist on the genome, and this might be a serious problem for highly variable RNA viruses. Although more recently the convenient and less laborious real-time PCR methods have been developed (see below) and are mostly used today in clinical practice, nested PCR assays are still used due to their high sensitivity and robustness.

Gel-based PCR is a heterogeneous, relatively laborious, detection method. Furthermore, it only reflects the end point of the PCR and, for this reason, doesn’t allow the determination of the initial quantity of the detected material, e.g., determination of the viral load. Since it lacks specific markers for the targeted amplicon, unspecific amplification yielding similar product sizes may lead to false positive detection. Nested PCR has the further disadvantage of being prone to cross-contaminations since reaction tubes with potentially very high quantities of target DNA are opened between the two reactions. Many of these drawbacks were solved by the advent of real-time PCR [6]. With this technique, the PCR product is monitored in the course of the reaction using DNA-binding moieties that alter their fluorescence upon binding to the amplified DNA. This allows a closed tube, homogeneous assay format, which reduces the risk for cross-contamination and also removes the laborious gel electrophoresis step. In addition, the cycle number where the fluorescence reaches a defined threshold level will depend on the initial quantity of target DNA or RNA (before reverse transcription).

Three main approaches have been taken to monitor fluorescence alteration in real time due to the buildup of the PCR product, which can be ordered according to the level of specificity the methods provide. The simplest method is to add a fluorescent dye to the PCR mixture with the property that the fluorescence intensity changes upon DNA binding. Typical dyes are asymmetric cyanine dyes, such as SYBR green or tiazole orange, that exhibit a fluorescence increase when bound to DNA [7, 8]. These types of real-time PCR have no better specificity than gel-based PCR, rather the opposite, since no information is provided about the product length. New possibilities are given by tethering the dye to one of the PCR primers that are constructed so that incorporation of the primer into the amplicon leads to an alteration of dye fluorescence. Several chemistries have been devised to this end, for example, scorpion primers [9], LUX primers [10], and Plexor primers [11]. Although in principle not providing a better specificity in regard to spurious amplification than the pure dye approach, fluorescent primers enable multiplexing by co-adding several primer pairs, each with a distinct fluorophore. The third approach includes addition of a third fluorescently labeled oligonucleotide, located between the primers, called a probe. The probe can also be labeled with a quencher (dual-labeled probe) but not always, e.g., not for the LightUp probes [7] or in the PriProET approach [12, 13]. Prominent examples of methods based on dual-labeled probes include TaqMan [14] and molecular beacons [15].

The signal that can be obtained from a probe-based real-time PCR experiment is often limited by the competing reannealing of the double-stranded PCR amplicon. Asymmetric PCR can be used to overcome this problem since it allows preferential amplification of one strand in a double-stranded DNA template. This is achieved by manipulating primer properties, most critically concentration, as well as other factors influencing primer melting temperature, such as length and nucleotide sequence. In the LATE-PCR method [16], asymmetric PCR has been combined with molecular beacons for readout to achieve a detection format that allows quantification from the end-point fluorescence. This format is suitable for simpler portable PCR instruments designed for detection in the field and has recently been commercialized by various companies.

The application of real-time PCR techniques and other methods in molecular diagnostics in veterinary medicine have recently been extensively reviewed [17, 18] and will be further discussed later in this section. To conclude this subsection, it is suitable to mention a recently developed method for the rapid molecular pathotyping of avian influenza [19] and Newcastle disease [20] viruses that combines several of the themes discussed here. This technique employs a three level semi-nested PCR format that utilize Plexor [11] fluorogenic primers as a detection mechanism. Furthermore, the assay format allows a highly multiplex interrogation of the sample by using primers in two vastly different concentration regimes. Instead of, as hitherto has been the case, requiring nucleotide sequencing over the hemagglutinin and fusion protein genes of avian influenza and Newcastle disease viruses, a much faster diagnosis can be obtained by a simple PCR-based method. This method could even be implemented on field PCR instruments for rapid on-site diagnosis and thereby providing means for faster containment of disease outbreaks.

With the development of DNA macro- and microarray technologies, it became possible to detect and characterize a wide variety of bacteria and viruses through simultaneous hybridization against large numbers of DNA probes immobilized on a solid support [35, 36].

The probes represent known sequences that may serve as markers for identification and/or genotyping of bacterial strains, resistance genes, viruses, etc. These are commonly arranged in an ordered array of spots (or features), and hybridization with a labeled target, i.e., the sample to be investigated, will therefore result in a hybridization profile in which individual probe results also can be assessed. As the names imply, the main difference between macro- and microarrays is the number and size of spots on the support. Macroarrays typically have larger and fewer spots and have proven particularly effective for detecting smaller subsets of genes, such as genes involved in antibiotic resistance [37]. Microarrays can contain thousands, and even up to many hundred thousands, of spots with different oligonucleotide probes and have successfully been used for detection and genotyping of bacterial and viral pathogens [38, 39]. The main advantages of microarray technology are high throughput, parallelism, miniaturization, and speed. However, microarrays are still considered to be an expensive technology and usually require large amounts of nucleic acid targets. Furthermore, unless it has been completely automated, the data analysis procedure might be time-consuming, and the results can be difficult to translate into information that is clearly communicable and decision supportive.

Genomic partitioning refers to the methodologies used for capture and enrichment of target regions. Within these methodologies, PLP has been used repeatedly for genotyping, localization, and array-based diagnostics. The earliest version of PLP consisted of two oligonucleotide probes of 20 nt connected by a linker region of 40 nt [33]. As the probes hybridize towards the target, the construct is ligated into a circular detector that can be replicated isothermally by Phi29 polymerase [40]. The detection can then be performed through incorporation of fluorophore tagged nucleotides. The PLP concept was further expanded with the introduction of the molecular inversion probe (MIP) technology. Where PLP leaves no gap after hybridization to the target region, MIP aims at leaving a single nucleotide gap. This gap is then filled in by addition of a single type of nucleotide into the assay. This approach enables substitutions on nucleotide level to be detected using just four reactions easily set up in a normal lab environment. It also provides a possibility of highly multiplexed designs of assays [41, 42]. Building on the same principle as PLP and MIP, the connector inversion probe (CIPer) technology extends the gap up to a few hundred nucleotides. Using DNA polymerase to fill the gap generates a product that can be sequenced, revealing the content of the target region [43]. Applications of PLP methodology and its derivatives for infectious diseases in animals include detection of all hemagglutinin and neuraminidase subtypes of AIV [44], as well as multiplex detection of FMDV, swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV) [45]. In addition, by designing different probes for the genomic and replicative form of the virus, it is possible to not only detect a virus but also localize it in relation to the host cells and perform semiquantitative analysis of the amount of replicative viruses, as demonstrated with porcine circovirus type 2 [46].

Although PLA is designed for detection of protein interactions and localization using antibodies for target recognition, hybridization events are required to generate a detectable signal [34]. Two sets of antibodies are designed: one targets the protein/s of interest and the other target the first set. The antibodies in the second set carry short oligonucleotide strands that can hybridize with special connector oligonucleotides and thereby enable the formation of circular DNA constructs. These are amplified and detected by PCR and fluorescent probes. The methodology combines dual antibody specificity with the signal amplification power of DNA amplification to produce a versatile and sensitive method for detection of very low amounts of targets. It also enables in situ localization studies of protein targets within cells [47]. Furthermore, PLA requires little to no sample preparation, making it ideally suitable for screening of massive amounts of samples, and can be used with a solid support to capture antigens for detection, similar to ELISA. The use of a solid support may also facilitate the removal of contaminants from the sample, thereby enabling PCR-based detection without the problem of inhibition. By combining the solid-support approach with RT-PCR detection, great sensitivity was demonstrated in a study of avian influenza virus [48]. Other applications of PLA technology include detection of several viruses, among them FMDV, with detection levels close to those of RT-PCR and 100-fold more sensitive than ELISA [49], as well as localization of influenza virus proteins within cells [50].