key: cord-0005364-gwkr4r1k
authors: Pfeffer, M.; Wiedmann, M.; Batt, C. A.
title: Applications of DNA amplification techniques in veterinary diagnostics
date: 1995
journal: Vet Res Commun
DOI: 10.1007/bf01839319
sha: 9282b6f2a3a4c79ab56505e5c040a8b94b0750de
doc_id: 5364
cord_uid: gwkr4r1k

An overview of the principles of the polymerase chain reaction, ligase chain reaction, self-sustained sequence replication and Qβ replicase is given. The application of these methods for the diagnosis of veterinary infectious and hereditary diseases as well as for other diagnostic purposes is discussed and comprehensive tables of reported assays are provided. Specific areas where these DNA-based amplification methods provide substantial advantages over traditional approaches are also highlighted. With regard to PCR-based assays for the detection of viral pathogens, this article is an update of a previous review by Belák and Ballagi-Pordány (1993).

The polymerase chain reaction (PCR) is an in vitro enzymatic method which allows several million-fold amplification of a specific DNA sequence. Since its introduction in 1985, PCR has facilitated the development of a variety of nucleic acid-based detection systems for bacterial, viral and other pathogens, as well as for genetic disorders (Erlich et al., 1991) . Owing to its high sensitivity, specificity and speed, PCR offers advantages over conventional diagnostic methods. While a variety of PCR-based assays have been described in the literature for the detection of infectious agents affecting man and animals, their application is not yet routine, even in large diagnostic laboratories. Problems with contamination, as well as cost-and time-intensive post-PCR detection methods, currently hamper its widespread use.

However, despite its current limitations, PCR already fmds relatively broad use in the routine diagnosis of hereditary diseases in animals, such as bovine leukocyte adhesion deficiency (BLAD) and porcine malignant hyperthermia syndrome (see Shuster et aL, 1992) .

Other recently developed DNA amplification methods, such as the ligase chain reaction (LCR), self-sustained sequence replication (3SR) and Qfl replicase amplification have so far found only limited applications, but show promise for specific diagnostic applications. While PCR and LCR rely on temperature cycling and therefore require some investment in the appropriate equipment, 3SR and Qfl replicase amplification are isothermal methods of DNA amplification, so making potential 'field' use feasible.

This review gives a short overview of the principles of the different DNA amplification techniques, with emphasis on current technical developments which will facilitate their use in veterinary diagnostics in the near future. Examples of diagnostic tests which show promise for more widespread application are also given.

The DNA amplification methods will be described briefly. More detailed reviews on the principles and technical details of the different methods can be found in Barany (1991) , Erlich and colleagues (1991) , Fahy and colleagues (1991) , Wolcott (1992) , Abramson and Meyers (1993) and Wiedmann and colleagues (1994b) .

PCR is defined by repetitive cycles, each consisting of three steps performed at different temperatures. In the first step, the double-stranded target DNA is denatured at high temperatures, resulting in single-stranded molecules. Two oligonucleotide primers each hybridize to their respective complementary DNA strands in the second step (annealing step), thus defining a region of the target DNA. In the last step, the 3' ends of these bound primers are extended by a thermostable DNA polymerase. During each cycle, the complementary DNA strands are copied by the sequential elongation of the two primers (see Figure 1 ). Newly synthesized DNA molecules can serve as templates in the next cycle, thus resulting in exponential amplification. A variety of parameters can affect the reaction kinetics and the success of amplification (for details see Xu and Larzul, 1991) ,

The specificity of the amplification depends on the primer design. Mispriming can be minimized by using a 'hot-start', whereby an essential PCR component (usually Taq polymerase) is added only after the annealing temperature is reached (Chou et al., 1992) .

The most common source of false positive results is carry-over contamination from previous PCRs (Kwok and Higushi, 1989) . A variety of methods have been developed to minimize carry-over contamination, which currently seems to be one of the major problems for the application of PCR in a routine diagnostic setting (Kwok, 1990) .

Amplification of RNA sequences, e.g. RNA viruses or mRNAs, requires transcription into cDNA using reverse transcriptase (RT) . In order to avoid additional manipulations and to diminish the risk of contamination, a single-tube RT-PCR format has been developed (Sellner et al., 1992) . Some thermostable DNA polymerases possess RT activity, so making a single-tube RT-PCR more feasible, allowing reduced manipulation time and minimizing carry-over contamination. Moreover, since reverse transcription is carried out at higher temperatures, longer cDNA molecules can be made, especially from RNAs with extensive secondary structures (Myers et al., 1994) . Figure 2 . A schematic representation of the ligase chain reaction (adapted from Barany, 1991) . Template DNA is shown by solid lines and the four primers are represented by hatched boxes. The site of the discriminating nucleotide is shown as a gap between one primer pair at the annealing step. Each primer pair can only be ligated when the overlapping 3' end of the light hatched primers contains the matching complementary nucleotide to the template. One cycle, consisting of a denaturing, a primer annealing and a ligation step, is indicated by brackets. For details see the section in the text on the ligase chain reaction PCR amplification products, termed amplicons, can be detected in several ways. Verification of the amplicon size by agarose gel electrophoresis using a molecular weight standard is applied in most cases. The PCR amplicon can also be digested with restriction endonucleases to confirm internal restriction enzyme sites. PCR in combination with restriction fragment length polymorphism (RFLP) is often used to type a group of pathogens or to define different alleles in the detection of hereditary diseases. A more time-consuming method is Southern blotting in which, after gel electrophoresis and blotting on a membrane, the PCR product is hybridized with a specific probe to verify homology.

The principle of LCR is based in part upon the ligation of two adjacent oligonucleotide primers, which uniquely hybridize to one strand of the target DNA (see Figure 2 ). The junction of the two primers is usually positioned such that the nucleotide at the 3' end of the upstream primer coincides with a known single base pair difference in the target sequence. This single base pair difference may define two different alleles, species or other phenotypic differences. If the target nucleotide at that site complements the nucleotide at the 3' end of the upstream primer, the two adjoining primers can be covalently joined by the ligase. The unique feature of LCR is a second pair of primers, almost entirely complementary to the first pair, which are designed with the nucleotide at the 3' end of the upstream primer, denoting the sequence difference. In a cycling reaction, using a thermostable DNA ligase, both ligated products can then serve as templates for the next reaction cycle, leading to an exponential amplification, analogous to PCR. If there is a mismatch at the primer junction, this structure will not be recognized by the thermostable ligase and the primers will not be ligated. The absence of the ligated product therefore indicates at least a single base pair change in the target sequence (Barany, 1991; Wiedmann et al., 1994b) . LCR is often utilized in conjunction with primary PCR amplification. Such a PCR-coupled LCR combines the sensitivity of PCR with the specificity of LCR for detection of each possible single base pair change.

3SR allows an exponential amplification of either RNA or DNA molecules, which are defined by two specific DNA primers. These primers are complementary to the target at their 3' ends and incorporate a promoter recognized by T7 RNA polymerase at the 5' end. Amplification is performed at a constant temperature and, where RNA is the template, a RT first produces a complementary DNA strand. This newly synthesized strand will be copied again by the RT, resulting in two newly synthesized DNA strands with a T7 promoter sequence at the 5' end. Therefore, T7 RNA polymerase can initiate synthesis of multiple copies (10-1000 per cycle) of complementary RNA strands. These RNA molecules can then be reverse transcribed to produce more DNA molecules. Only in an RNA-DNA hybrid will the RNA strand then be degraded by a third enzyme, RNaseH. The DNA strands are therefore free to initiate more RNA synthesis, which ensures continuation of the amplification (Fahy et al., 1991) . A schematic diagram of this process is shown in Figure 3 .

1. c y c l e -2. c y c l e -< m p r i m e r a n n e a l i n g r e v e r s e t r a n s c r i p t i o n a n d R N a s e H d i g e s t i o n p r i m e r "~"~ a n n e a l i n g I p r i m e r a n n e a l i n g Q/3 replicase amplification utilizes the replicase from Q/~ bacteriophage, an RNA-dependent RNA polymerase, to self-replicate an RNA template designated MDV-1. In this assay, unlike in PCR, a reporter RNA rather than the actual target is amplified. Specificity is achieved by inserting a target-specific probe sequence in MDV-1. After hybridization of the probe-MDV-1 sequence to the target, unbound probe is removed and the remaining probe is amplified after addition of Q/~ replicase (see Figure 4 ). The resulting amplified probe-MDV-1 sequences can be visualized in ethidium bromide-stained gels or by use of a secondary probe complementary to MDV-1 sequences. An example of a sensitive non-isotopic Q/~ replicase assay for detecting an infectious agent (Chlamydia trachornatis) has recently been reported (Shah et aL, 1994) .

Both 3SR and QB replicase have the advantage of being isothermal, and therefore do not require thermal cyclers. This offers potential for use outside well-equipped laboratories and might make these procedures especially useful for some veterinary applications.

PCR has proved to be a very valuable technique for the detection of many infectious agents, among which viruses form the largest group. Table I lists PCR-based assays for the diagnosis of veterinary viral pathogens. Owing to the very large number of publications which appear every year, only those that have been published since the last review by Belfik and Ballagi-Pordfiny (1993) are included. The herpesviruses have been selected here as a relevant example of how DNA amplification-based assays can be applied to veterinary diagnostic problems.

Animal herpesviruses are known to cause severe losses in livestock. While the acute form of the diseases can easily be diagnosed by virus isolation, there is at present no direct method for detecting the latent state of infection. During latency, infectious virus cannot be isolated because only the herpesviral DNA persists within the cells and no viable virus is produced. In contrast to human herpesviruses, latency-associated transcripts have not been found in any of the animal herpesviruses, apart from pigs infected with pseudorabies virus (PRV; Cheung, 1989) . However, using PCR, the trigeminal ganglion has been shown to be one location for latent equine herpesvirus type 1 (EHV-1; Slater et aL, 1994) , for PRV in pigs (Bel~k et al., 1989) and for feline herpesvirus (FHV) in cats (Reubel et al., 1993) . The virus, however, can be reactivated by superinfection or immunosuppression. It is therefore of epidemiological importance to identify carriers in order to vaccinate or to cull them. (1994) During campaigns to eradicate animal herpesviruses, genetically altered modified live vaccines (so-called 'marker vaccines') have been employed to combat and displace wild-type (wt) viruses. Vaccine strains of PRV have been generated that lack one of the four non-essential glycoproteins, i.e. gp63 (Petrovski et al., 1986) , gI (Quint et al., 1987) , glII (Kit et aL, 1987) , and gX (Marchioli et aL, 1987) . ELISAs with monoclonal antibodies, which distinguish between vet and vaccine virus, were used for screening wt PRV carriers, but gave some false positive results (Annelli et aL, 1991) . Consequently, a panel of PCR-based assays has been developed to replace these ELISAs and to minimize false positives (see Bel~k and Ballagi-Pord~ny, 1993 ; and Table I ). Recently, a BHV-1 gE deletion mutant has been developed as a potential modified live vaccine (Kaashoek et al., 1994) . PCR would be suitable to screen for wt BHV-1 carriers in BHV-1 eradication programmes. A deletion mutant of EHV-1 (strain RacH) is widely used as a vaccine strain in Europe. Owing to the lack of monoclonal antibodies which discriminate between the vaccine and wt EHV-1 strains, RacH-induced abortions of vaccinated mares could not be excluded. To allow specific screening for the vaccine strain, a PCR assay which discriminates RacH from EHV-1 and EHV-4 field strains has been developed (Osterrieder et aL, 1994) .

PCR has been successfully applied to the detection of EHV-1 and EHV-4 in nasal swabs (Sharma et aL, 1992; Gilkerson et aL, 1994) and in aborted fetuses, replacing time-consuming virus isolation and immunofluorescence detection methods (Hardt et al., 1992; Borchers and Slater, 1993; Kirisawa et aL, 1993; Osterrieder et aL, 1994) . PCR assays have also been developed to detect bovine herpesvirus type 1 (BHV-1; e.g. Vilcek, 1993) , type 3 (BHV-3, formerly named BHV-4; e.g. Naeem et al., 1991) and ovine herpesvirus type 2 (OHV-2; Wiyono et al., 1994) . Direct detection by PCR of BHV-1 from nasal swabs from cattle showing respiratory tract diseases has also been described (van Engelenburg et aL, 1993; Vilcek et aL, 1994b) .

Direct diagnosis of herpesviruses by virus isolation from some biological materials, such as semen, has proved to be difficult owing to cytotoxic components which interfere with tissue culture tests. Nevertheless, to control the transmission and spread of BHV-1 through semen, practicable PCR protocols have been developed for amplification of BHV-1 sequences (van Engelenburg et aL, 1993; Wiedmann et aL, 1993b) .

Methods for isolating DNA or RNA from fixed tissues have been improved (Kallio et aL, 1991; Koopmans et al., 1993) . While viral proteins are usually no longer immunologically detectable and viable virus cannot be isolated from formalin-fixed material, PCR often allows a retrospective diagnosis of viral infections using such pathological specimens (Rimstad and Evensen, 1993; Osterrieder et aL, 1994) .

Overviews of PCR and other DNA amplification-based assays for detecting veterinary bacterial and fungal pathogens are given in Tables II and III, ( Nascimento et al. (1991) ; Kempf et al. (1993 Kempf et al. ( , 1994b Dussurget and Roulland (1994); Rawadi et al. (1993) Kempf et aL ( aAll the PCR assays listed detect one or more pathogenic Leptospira spp., some of them are specific for a certain serovar; for more details on the specificity of the particular PCR refer to the respective reference Detection systems for pathogenic bacteria and fungi based on P C R or other D N A amplification techniques usually depend upon the availability of well characterized, genus-or species-specific target sequences. This strategy is easily applied to well-documented bacterial and fungal pathogens, where the sequence of one or m o r e genes is known (e.g . Listeria m o n o c y t o g e n e s , S a l m o n e l l a spp,) . However, for many animal pathogens there is not sufficient information available for the design of species-specific PCR primers. The 16S rRNA gene, encoding part of the prokaryotic rRNA, consists of both highly conserved and variable regions. The latter regions usually contain at least single base pair differences that are species-specific or longer stretches of genus-specific differences. A general method for PCR amplification and sequencing of this gene has been described by Weisburg and colleagues (1991) . Other methods, such as direct sequencing of the 16S rRNA using reverse transcriptase, are also available, although this technique does not always seem to be sufficiently accurate (Collins et al., 1991) , After determination of the 16S rRNA gene nucleotide sequence for the organism of interest, species-or genus-specific sequences can be determined by alignment with sequences present in the 16S rRNA database and, where necessary, by sequencing the 16S rRNA for closely related species. Currently, 16S rRNA sequence data are available for more than 1500 bacterial species (Olsen et al., 1994) . (1994) While the design of genus-specific PCR primers based on 16S rRNA sequences is relatively easy (see Bleumink-Phiym et al., 1994; , the development of species-specific assays is usually more difficult. In many cases, closely related species within the same genus differ only by a single base pair difference in one of the variable regions of the 16S rRNA. Restriction fragment length polymorphism (RFLP) of a PCR amplicon offers one possibility for discriminating these single base pair differences, but only if a suitable restriction site is present. PCR-RFLP is, however, a fairly cumbersome technique which does not lend itself to automation. PCR assays for discriminating single base pair differences have also been described, but such an allele-specific PCR often does not allow reliable discrimination of any single base pair difference . A different approach to achieving specific detection of a bacterial pathogen based on species-specific 16S rRNA sequences has been reported by Wiedmann and colleagues (1992) . After initial sequencing of the 16S rRNA genes of various L. monocytogenes, and of the closely related non-pathogenic bacterium L. innocua, consistent single base pair differences specific for L. monocytogenes were located (Czajka et aL, 1993) . These sequences were used to design LCR primers able to specifically identify L. monocytogenes. To improve the sensitivity of this LCR, a set of flanking PCR primers was employed to initially amplify the segment containing the specific single base pair difference (Wiedmann et al., 1992) . This PCR-coupled LCR was shown to be highly specific for L. monocytogenes and was able to detect a minimum of 10 colony-forming units using a non-isotopic detection method (Wiedmann et al., 1993a) .

DNA amplification-based systems can be used simply to detect a bacterial species, as well as to obtain information about the characteristics of these bacteria. For example, in human medical diagnostics, PCR probes have been developed to detect Staphylococcus aureus using 16S rRNA or gyrA primers and to assess methicillin resistance (Geha et aL, 1994; Zambardi et al., 1994) . These probes can be employed in a multiplex PCR to identify methicillin resistant and sensitive S. aureus strains in a single PCR reaction.

The differentiation of vaccine strains from wild-type strains of the same species can also be achieved using PCR. An example is the detection of the Mycoplasma gallisepticum F-vaccine strain using primers for fMGF-1 (Nascimento et aL, 1993) .

Suitable target sequences for the detection of fungal pathogens are often more difficult to define than those for bacterial pathogens. The characterization of virulence genes in fungi is more demanding, since tools to analyse their genetics are less advanced and their genetic structure is more complex. However, for fungi, cloning and sequencing of the rRNA genes (5S, 5.8S, 18S and 23S), their internal transcribed spacers and non-transcribed spacers can be achieved using PCR primers targeting conserved regions. This genetic information can then be used to achieve a genus-or species-specific detection system analogous to the system described above for the detection of specific 16S rRNA genes in bacteria (see Check, 1994) .

The detection of bacterial and fungal pathogens using DNA amplification-based techniques leads to results which have to be interpreted differently from those obtained using cultural methods. PCR and other similar techniques detect DNA from the targeted organism, whether this organism is alive or not. This is of particular concern with regard to the direct detection of microorganisms from environmental samples, where organisms killed by disinfectants might still give positive results by PCR. The detection of non-viable bacteria might, however, offer an advantage over cultural methods if animals treated with antibacterial or antifungal substances are to be tested. In such cases, DNA amplification-based methods might allow diagnosis 'after the fact', where cultural methods would give negative results.

The application of DNA amplification methods for diagnosing parasitic infections is still limited, for various reasons. Diagnosis of ectoparasitic diseases can often be achieved by clinical examination of the animal and, in most cases, the parasite can be identified macroscopically or microscopically. Therefore, there is no substantial need for PCR or other DNA amplification methods as a diagnostic tool for ectoparasites. However, in two areas ampfification methods are applicable to ectoparasites. PCR has been applied to detect veterinary pathogens carried by these parasites. Examples include the detection of Borrelia burgdorferi, the agent of Lyme disease, in Ixodes ticks Roos and Grant (1993) (Persing, 1991) and of arthropod-borne viruses, which are transmitted by mosquitoes, ticks and other blood-feeding arthropods (Ward et aL, 1990; Vodkin et al., 1993 Vodkin et al., , 1994 . PCR allows easy screening of large numbers of samples and is therefore helpful for detecting infected vectors in epidemiological surveys. Another application for PCR-based techniques is the determination of phylogenetic relationships among species of parasites, which enables reassessment of their current taxonomic classification (Brindley et aL, 1993) . Owing to the complexity of these organisms and the lack of sufficient sequence information, modified PCR techniques have been applied for DNA fingerprint analysis in parasites. These include amplification of minisatellite repeats (MVR-PCR; for review see Arnot et aL, 1994), random amplified polymorphic DNA (RAPD; MacPherson and Gajadhar, 1993; Dupouy-Camet et al., 1994) or the amplified polymorphic-PCR (AP-PCR; McClelland and Welsh, 1994) . PCR-based fingerprinting techniques have also been applied to helminths and protozoan parasites (see Table IV ). An increasing number of purely diagnostic applications of PCR techniques have been described, mostly for protozoan parasites (Wilson, 1991; van Eys et aL, 1992; Webster et al., 1993; Awad-E1-Kariem et al., 1994; Figueroa et aL, 1994; Howe and Sibley, 1994; Majiwa et aL, 1994) . Because of the intracellular localization of most protozoans, accurate identification of the infective agent is difficult by conventional methods. Molecular approaches, such as DNA hybridization assays, have been applied, but they are sometimes not sufficiently sensitive (MacPherson and Gajadhar, 1993) . Conversely, the sensitivity of a diagnostic test is not an important issue for a wide variety of diarrhoea-causing protozoans, because they are usually shed in high numbers. Rather, rapid and convenient identification of different Coccidia spp. often proves difficult because unequivocal morphological markers are missing (Brindley et al., 1993) . The identification of more virulence genes and of specific DNA markers for all kinds of pathogenic parasites will probably facilitate the development of further DNA-based assays for veterinary parasites in the near future. This has been done for human parasites, e.g. Plasmodia, Trypanosoma and Leishmania. Nevertheless, application of such assays will only be important for routine diagnostic purposes where conventional methods cannot provide either the necessary sensitivity or a quick and reliable identification of the species.

Over the last few years, a variety of hereditary diseases in animals have been traced back to the respective genes and mutations responsible for the biochemical defects and clinical syndromes. PCR facilitated the use of this information for rapid diagnostic assays which allow the screening of large numbers of animals. The majority of hereditary diseases are caused by single base pair mutations, which in many cases result in the loss or acquisition of a restriction enzyme recognition site. Most assays for the detection of these single base pair mutations are based on primary PCR amplification of a region containing the polymorphic site, followed by a restriction digest of the PCR product with a suitable restriction enzyme, such as PCR-RFLP. Current assays for BLAD, citrullinaemia and hyperkalaemic periodic paralysis provide examples of this type of assay (see Table V ). For some hereditary diseases, routine diagnosis now involves DNA sequencing after primary PCR ampfification of mRNA or of the afflicted exon (Zheng et aL, 1994) . This approach is usually applied where no specific single base pair change can be linked to a hereditary condition or where no change in a restriction site coincides with a given mutation (Zheng et aL, 1994) .

While PCR-RFLP provides a good diagnostic system for detecting carriers of many disease alleles, this system has two major disadvantages. First, not all potential single base pair mutations lead to a change in a restriction site. Second, although screening of large numbers of animals can be performed by PCR-RFLP, the technique is very cumbersome and time-consuming and cannot easily be integrated into an automated format. Recently, a non-isotopic LCR assay has been described for the detection of BLAD which overcomes these disadvantages (Batt et aL, 1994) .

A list of DNA amplification-based assays for the detection of hereditary diseases is given in Table V .

The application of DNA amplification methods is not limited to the diagnosis of infectious or hereditary diseases. This section gives an overview of other applications in veterinary diagnostics, excluding purely research-oriented areas, such as the detection of mRNA for cytokines or other immunomediators.

PCR or PCR-RFLP analysis is often used in animal breeding to determine the genotype of animals for specific production traits, e.g. the genotype for certain milk proteins. Examples include typing for fi-lactoglobulin alleles in sheep (Schlee et aL, 1993) or for a-Sl-casein, fi-casein, x-casein, fl-lactoglobulin or a-lactalbumin alleles in cattle (Pinder et aL, 1991; Schlieben et al., 1991; Sulimova et al., 1991; David and Deutch, 1992; Rottmann and Schlee, 1992; Schlee and Rottmann, 1992; Wilkins and Kuys, 1992) . Another example of a PCR application in animal breeding is an assay for the sex-linked late-feathering gene in chickens (Iraqi and Smith, 1994) .

Only the advent of PCR allowed preimplantation sex determination in embryo transfer. Usually, male sex is determined using primers specific for the sex-determining region on the Y chromosome (SRY). Such PCR assays have been described for cattle, goats, sheep, pigs and a variety of other animals (Miller, 1991; Bredbacka and Peippo, 1992; Kageyama et al., 1992; Rao and Totey, 1992; Utsumi et 

Brening & Brem (1992) ; Brem & Brening (1993); Otsu et al. (1992) al., 1992; Horvat et al., 1993; Saitoh and Totsukawa, 1993; Kawarasaki et al., 1994) . Recently, a multiplex PCR has been described which can be used to screen bovine preimplanted embryos for sex and four genetic diseases (Schwerin et al., 1994) . PCR has also been used to prescreen microinjected bovine embryos for the presence of a transgene construct (Horvat et aL, 1993) . Another application of PCR with male sex-specific primers is the determination of bovine chimerism in female calves co-twin to a male (so-called 'freemartins') (Grobet et aL, 1992; Lipkin et al., 1993) . Forensic veterinary applications of DNA amplification-based systems include the species determination of meat and carcasses. Species-specific primers have been described for sheep, goats and cattle (Chikuni et al., 1994; Wagner et al., 1994) . Species-specific primers for the amplification of regions containing a variable number of tandem repeats have been used to determine parentage in most domestic animal species (Buitkamp et al., 1994) .

Nucleic acid-based technology will have a large impact on the diagnosis and monitoring of many animal diseases. High demands have been placed on DNA amplification techniques. With regard to speed, reliability and cost, these techniques, and in particular PCR, are often superior to conventional diagnostic methods. However, in contrast to ELISA systems, there are only a few PCR kits for human or veterinary pathogens commercially available.

A patent covering the PCR process and patents for specific products, such as purified thermostable enzymes, are held by Hoffman-LaRoche. This means that the user of the PCR process needs to be properly licensed. For research and development applications, PCR can be performed if licensed enzymes and licensed instruments are used. In contrast, when a PCR assay is to be routinely used in commercial veterinary diagnostics, an end-user 'service license' has to be acquired. The contract with Hoffman-LaRoche includes a royalty obligation and enables the licensee to perform any kind of veterinary diagnostic testing. The royalty rate varies between 5% and 15%, depending on the application (A. Junosza-Jankowski, PCR Licensing Manager, Roche Diagnostic Systems, personal communication, 1995) .

In veterinary diagnostics, the application of PCR is currently restricted to fully equipped diagnostic laboratories. Its implementation by veterinary practitioners under field conditions is not straightforward, but nor is it insurmountable (Barker, 1994) . Recent developments allow the detection of amplification products by a system which uses microtitre plates. Labelling of the primers with digoxigenin, biotin or different fluorogens has been successfully applied in LCR and PCR (e.g. Wiedmann et al., 1993a) . While the ligand group is captured by an immobilized 'receptor' (e.g. biotin-streptavidin), the reporter group on the other end of the amplicon can be used for the detection. Other approaches use microtitre plates coated with a probe to capture the PCR product, which is then detected by a second probe. Very recently, a new approach for the detection and quantification of PCR products has become commercially available. Taqman TM (Perkin Elmer/Applied Biosystems Division) uses a probe which is located between the PCR primers. This probe is labelled with a fluorescent reporter and quencher dye. The fluorescence emitted by the reporter dye is normally quenched by the quencher dye but increases upon hydrolysis of the probe, an event that only occurs during amplification of the target DNA. This system has the potential to allow detection and quantification of PCR products in a microtitre plate in less than 10 minutes (Lee et aL, 1993; Anonymous, 1994) .

We believe that DNA amplification-based methods will replace conventional methods in some fields of diagnostics, such as hereditary diseases where the dysfunctional genes are known and viral diseases of persistently infected animals. In other areas, DNA amplification methods will be used to complement conventional diagnostics. Meyers, T.W., 1993. Nucleic acid amplification technologies. Current Opinion in Biotechnology, 4, 41-47 Agresti, A., Ponti, W., Rocchi, M., Meneveri, R., Marozzi, A., Cavalleri, D., Peri, E., Poli, G. and Ginelli, E., 1993 

Development and evaluation of PCR test for detection of Taylorella equigenitalis

A nested PCR for the detection and differentiation of EHV-1 and EHV-4

Rapid discrimination of Echinococcus species and strains using a polymerase chain reaction-based RFLP method

Cattle strain of Echinococcus granulasus and human infection

Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene

Sex diagnosis of ovine and bovine embryos by enzymatic amplification and digestion of DNA from the zfy-zfx locus

Use of molecular genetic diagnosis of malignant hyperthemaic syndrome (MHS) in selection of pigs

Genomic organization and analysis of the 5' end of the porcine ryanodine receptor gene ryrl. FEBS (Federation of

Detection of Echinococcus multilocularis DNA in fox faeces using DNA amplification

Differentiation of Toxoplasma gondii from closely related coceidia by riboprint analysis and a surface antigen gene polymerase chain reaction

The use of PCR genome mapping for the characterization of TGEV strains

Host-virus interaction as defined by amplification of viral DNA and serology in lentivirus-infected sheep

Maternal factors associated with parental transmission of ovine lentivirus

Use of the polymerase chain reaction (PCR) to detect DNA from Renibacterium salmoninarum within individual salmonid eggs

Bovine rotavirus V1005 a P5, not a P12, type like all viruses in a German survey

A combined modified reverse dot blot and nested PCR assay for the specific nonradioactive detection of Listeria monocytogenes

Genetisches Fingerabdruekverfahren und 'DNA-Profil' bei Haustieren

Long-term persistent infection of swine monocytes/macrophages with African swine fever virus

A rapid method for the extraction and detection of Mycobacterium avium subspecies paratuberculosis from clinical specimens

Molecular techniques shed light on fungal genetics

Detection of pseudorabies virus transcripts in trigeminal ganglia of latently infected swine

Polymerase chain reaction assay for detection of sheep and goat meats

Prevention of pre-PCR mis-prinaing and primer dimerization improves low-copy-number amplification

Detection of Salmonella enteritidis in feces from poultry using booster polymerase chain reaction and oligonucleotide primers specific for all members of the genus Salmonella

Genus-specific detection of salmonellae in equine feces by use of the polymerase chain reaction

Multiplex polymerase chain reaction based assay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood

Polymerase chain reaction-based diagnostic assay to detect cattle chronically infected with Babesia boris

Multiplex PCR for identification of methicillin-resistant staphylococci in the clinical laboratory

Sample preparation method for polymerase chain reaction-based semiquantitative detection of Leptospira interrogans serovar hardjo subtype hardjobovis in bovine urine

Epidemiological investigation of equid herpesvirus-4 (EHV-4) excretion assessed by nasal swabs taken from thoroughbred foals

Isolation of pseudorabies (Aujeszky's disease) virus from a Florida panther

Diagnostic-identification of Taenia saginata with the polymerase chain reaction

Use of polymerase chain reaction to detect porcine parvovirus associated with swine embryos

Detection of verotoxigen~c Escherichia coli in bull semen using the polymerase chain reaction

Nucleotide sequences of Australian isolates of the feline immunodeficiency virus: comparison with other feline lentiviruses

D6tection du freemartinisme ~ l'aide d'empreintes gEn6tiques et d'une sonde Y-sp6cifique bovine

Detection of bovine viral diarrhea virus RNA in formalin-fixed, paraffin-embedded brain tissue by nested polynaerase chain reaction

Detection of the pathogenic parasite Toxoplasma gondii by specific amplification of ribosomal sequences using comultiplex polymerase chain reaction

Detection ofAeromonas salmonicida from fish by using polynlaerase chain reaction amplification of the virulence surface array protein gene

Reverse transcriptase-PCR assay for detection of hog cholera virus

Die Polymerasekettenreaktion (PCR) zum Nachweis yon DNA der Equinen Herpesviren 1 und 4. Berliner und Muenchner Tieraerztliche Wochenschrifl

Development of gene probes for the specific identification of Streptococcus uberis and Streptococcus parauberis based upon large subunit rRNA gene sequences

Gene specific assay to differentiate strains of pseudorabies virus

Comparison of the nucleotide sequence and development of a PCR test for the epsilon toxin gene of Clostridium perfringens type b and type d

DNA probe for A eromonas salmonicida

Detection of bovine trichomoniasis with a specific DNA probe and PCR amplification system

Detection of foot-and-mouth disease virus RNA in clinical samples and cell culture isolates by amplification of the capsid coding region

A hemi-nested PCR assay for the detection and identification of vesicular stomatitis virus nucleic acid

Detection of Candida albicans and other yeasts in blood by PCR

Sexing and detection of gene constuct in microinjected bovine blastocysts using the polymerase chain reaction

Detection of Mycoplasma boris using in vitro deoxyribonucleic acid amplification. Revue Scientifique et Technique de l

Toxoplasma gondii: Analysis of different laboratory stocks of the RH strain reveals genetic heterogeneity

Application of the polymerase chain reaction for the detection of Ehrlichia canis in tissues of dogs

Comparison of PCR and other tests for early diagnosis of canine ehrlichiosis

Determination of the zygosity of ev21-K in late-feathering male White Leghorns using the polymerase chain reaction

Determination of bovine rotavirus G and P serotypes by polymerase chain reaction

Feline leukemia virus detection by imnmnohistochemistry and polymerase chain reaction in formalin-fixed, paraffin-embedded tumor tissues from cats with lymphosarcoma

Development of polyanerase chain reaction assays for detection of Listeria monocytogenes in clinical cerebrospinal fluid samples

A conventionally attenuated glycoprotein E-negative strain of bovine herpesvirus type 1 is an efficacious and safe vaccine

Amplification and sequence analysis of sty sex-determining region y conserved region of domestic animals using polDnerase chain reaction

A simple method for isolation of DNA from formalin-fixed paraffin-embedded samples for PCR

Diagnosis of rabies by polymerase chain reaction with nested primers

Colorimetric diagnosis of prolonged bluetongue viremia in sheep, using an enzyme-linked oligonucleotide sorbent assay of anaplified viral nucleic acids

Presumptive diagnostic differentiation of hog cholera virus from bovine viral diarrhea and border disease viruses by using a cDNA nested-amplification approach

Detection of porcine male-specific DNA sequence using polymerase chain reaction: Effect of template cell number and amplification conditions

Detection of leptospiral DNA by PCR

Early detection of bovine leukemia virus in cattle by use of the polymerase chain reaction

The polymerase chain reaction for Mycoplasma gallisepticum detection

Comparison of antigenic and pathogenic properties ofMycoplasma iowae strains and development of a PCR-based detection assay

Mycoplasma gallisepticum infection in drug-treated chickens: comparison of diagnosis methods including polymerase chain reaction

Sequence conservation in the RNA polylnerase gene of infectious bursal disease virus

Detection and identification of equine herpesvirus-1 and -4 by polymerase chain reaction

Second-generation pseudorabies vaccine with deletions in thymidine kinase and glycoprotein genes

Bovine leukaemia virus: Rapid detection of proviral DNA by nested PCR in blood and organs of experimentally infected calves

Optimization of extraction and PCR anaplification of RNA extracts from paraffin-embedded tissues in different fixatives

Highly sensitive non-isotopic DNA hybridization system using anaplification polymerase chain reaction for identification and indication of Brucella

Differentiation of two rabies strains in Estonia with reference to recent Finnish isolates

Procedures to minimize PCR-product carry-over

Avoiding false positives with PCR

Effects of primer-template mismatches on the polymerase chain reaction: human immunodefMency virus type 1 model studies

Differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction fragment length polymorphism analysis

Polymerase chain reaction and a biotin-labeled DNA probe for detection of infectious bronchitis virus in chickens

Detection of Dichelobacter nodosus using species-specific oligonucleotides as PCR primers

Development and application of a polymerase chain reaction assay for Mycoplasma synoviae

The detection and quantification of feline immunodeficiency provirus in peripheral blood mononuclear cells using the polymerase chain reaction

Allelic discrimination by nick-translation PCR with fluorogenic probes

A PCR-based assay for the identification of the fish pathogen Renibacterium salmoninarum

Detection of Pseudomonas pseudomallei by PCR and hybridization

Detection of feline coronaviruses in cell cultures and in fresh and fixed feline tissues using polymerase chain reaction

Sequence comparison of a highlyvirulent infectious bursal disease virus prevalent in Japan

Early PCR amplification test for identifying chimerism in female calves co-twin to a male in cattle

A detection method for infectious pancreatic necrosis virus (IPNV) based on reverse transcription (RT)-polymerase chain reaction (PCR)

DNA fingerprinting by arbitrarily primed PCR

Detection of bluetongue virus in the blood of inoculated calves: comparison of virus isolation, PCR assay, and in vitro feeding of Culicoides variipennis

Differentiation of seven Eimeria species by random amplified polymorphic DNA

Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish, detected by nested reverse transcription-PCR of 16S rRNA sequences

A cloned DNA probe for Cowdria ruminantium hybridizes with eight heartwater strains and detects infected sheep

A quantitative polymerase chain reaction method for the detection in avian faeces of salmonellas carrying the spvR gene

Detection of trypanosome infections in the saliva of tsetse flies and buffy-coat samples from antigenaemic but aparasitaemic cattle

Direct and rapid detection of Erysipelothrix rhusiopathiae DNA in animals by PCR

Detection ofBorrelia burgdorferi using the polymerase chain reaction

A vaccine strain of pseudorabies virus with deletions in the thymidine kinase and glycoprotein X genes

Detection of porcine reproductive and respiratory syndrome virus and efficient differentiation between Canadian and European strains by reverse transcription and PCR amplification

Sensitive detection of trypanosomes in tsetse flies by DNA amplification

Canine distemper virus transcripts detected in the bone cells of dogs with metaphyseal osteopathy

General primer-mediated PCR for detection of Aspergillus species

Architecture of the canine iduA gene and mutation underlying canine mucopolysaccharidosis I

Polymerase chain reaction for detection of Leptospira spp. in clinical samples

Sequence alterations within and downstream of the A-type inclusion protein genes allow differentiation of orthopoxvirus species by polymerase chain reaction

Isolation of Y chromosome-specific sequences and their use in embryo sexing

Detection of bovine leukemia virus proviral DNA in individual cells

Unique oligonucleotide primers in PCR for identification of Cryptococcus neoformans

Isolation and characterization of a species-specific DNA fragment for detection of Candida albicans by polymerase chain reaction

Comparison of polymerase chain reaction with virus isolation and haemagglutination assays for the detection of canine parvovirus in faecal specimens

Detection of feline immunodeficiency virus proviral DNA in feline peripheral blood mononuclear cells by the nested two-step pol3naaerase chain reaction

High temperature reverse transcription and PCR with Thermus thermophilus DNA polymerase

Identification of regional variants of the rabies virus within the Canadian province of Ontario

Detection of bovine immunodeficiency-like virus by the polymerase chain reaction

Tissue distribution of bovid herpesvirus-4 in inoculated rabbits and its detection by DNA hybridization and polymerase chain reaction

Differentiation of toxigenic from nontoxigenic isolates of PasteureUa multocida by PCR

Polymerase chain reaction for detection of Mycoplasma gallisepticum

Mycoplasma gallisepticum F-vaccine strain-specific polymerase chain reaction

Amplification of Salmonella chromosomal DNA using the polymerase chain reaction

Polymerase chain reaction (PCR) amplification of RNA of striped jack nervous necrosis virus (SJNNV)

Antigenic and genetic conservation of the haemagglutinin in H1NI swine influenza viruses

Characteristic differences in reverse transcripfion-polymerase chain reaction products of ovine, bovine and human respiratory syncytial viruses

Identifying bovine respiratory syncytial virus by reverse transcription-polymerase chain reaction and oligonucleotide hybridizations

Minireview: the winds of (evolutionary) change: breathing new life into microbiology

A touchdown PCR for the differentiation of equine herpesvirus type 1 (EI-IV-1) field strains from the modified live vaccine strain RacH

Refinement of diagnostic assays for a probable causal mutation for porcine and human malignant hyperthermia

Specific detection of Campylobacterjejuni and Campylobacter coli by using pol3maerase chain reaction

Nucleotide sequence of the splice junction of feline leukemia virus envelope lnRNA

Characterization of field strains of group A bovine rotaviruses by polymerase chain reacfion-generated G and P type-specific cDNA probes

Polymerase chain reaction: trenches to benches

Deletions in vaccine strains of pseudorabies virus and their effect on synthesis of glycoprotein gp63

A ligase chain reaction targeting two adjacent nucleotides allows the differentiation of cowpox virus from other orthopoxvirus species

Analysis of polymorphism in the bovine casein genes by use of the polymerase chain reaction

A polymerase chain reaction (PCR) protocol for the specific detection of Chlamydia spp

Detection of bovine leukemia virus RNA in serum using the polymerase chain reaction

Analysis of sites of foot and mouth disease virus persistence in carrier cattle via the polynaerase chain reaction

Construction and characterization of deletion mutants of pseudorabies virus: a new generation of 'live' vaccines

Sex determination in sheep and goats using bovine Y-chromosome specific primers via polymerase chain reaction: potential for embryo sexing

Detection and identification of Mycoplasma contamination in cell cultures by polymerase chain reaction

Cosegregation of codon 807 mutation of the canine rod cGMP phosphodiesterase 3 gene and rcdl

Molecular diagnostic tests for the ascertainment of genotype at the rod cone dysplasia 1 (rcdl) locus in Irish setters

Specific amplification of Aspergillus fumigatus DNA by polymerase chain reaction

Detection of caprine arthritis~encephalitis virus by polymerase chain reaction

Diagnostic evaluation of polymerase chain reaction discriminative for Bordetella pertussis, B. parapertussis and B. bronchiseptica. Diagnostic Microbiology and Infectious Disease

Detection of active and latent feline herpesvirus 1 infections using the polymerase chain reaction

The identification of equid herpesvirus 1 in paraffin-embedded tissues from aborted fetuses by polymerase chain reaction and immunohistochemistry

Delayed seroconversion following naturally acquired caprine arthritis-encephalitis virus infection in goats

Rapid detection of vesicular stomatitis virus New Jersey serotype in clinical samples by using polymerase chain reaction

Development of PCR method specific for Marek's disease virus

Species-specific PCR for the parasitic nematodes Haemonchus contortus and Trichostrongylus colubriformis

Characterization of the 16S rRNA genes from Mycoplasma sp. strain F38 and development of an identification system based on PCR

Analysis of milk protein genes with PCR-RFLP and PASA

Periodic paralysis in quarter horses: a sodium channel mutation disseminated by selective breeding

Detection of sly sex-determining region y conserved region in pig leukocytes using polymerase chain reaction

Molecular epidemiology of foot-and-mouth disease type O

Rapid detection of African horsesickness virus by the reverse transcriptase polymerase chain reaction (RT-PCR) using the amplimer for segment 3 (VP3 gene)

Mycobacterium paratuberculosis DNA in Crohn's disease tissue

Identification of Bordetella avium using the polymerase chain reaction

Detection and identification of Leptospira interrogans serovars by PCR coupled with restriction endonuelease analysis of amplified DNA

A quantitative technique for the study of the latency of Aujeszky virus

Genotyping of bovine beta casein, beta lactoglobulin and alpha lactalbumin using the polymerase chain reaction

Genotyping of ovine beta-lactoglobulin alleles A and B using the polymerase chain reaction

Genotyping of bovine kappa casein kappa-cn-a kappa-en-b kappa-cn-c kappa-cn-e following DNA sequence anaplification and direct sequencing of kappa-en-e PCR product

Evaluation of PCR for diagnosis of bovine viral diarrhea virus in tissue homogenates

Identification of the gene encoding the major capsid protein of fish lymphocystis disease virus

Differential diagnosis of infectious laryngotracheitis from other avian respiratory diseases by a simplified PCR procedure

DUMPS cattle carry a point mutation in the uridine monophosphate synthase gene

Simultaneous genetic typing at different loci in bovine embryos by multiplex polymerase chain reaction

Reverse transcriptase inhibits Taq polyaaaerase activity

Novel, ultrasensitive, Q-beta replicase-amplified hybridization assay for detection of Chlamydia trachomatis

Diagnosis of equid herpesviruses-1 and -4 by polymerase chain reaction

Identification and prevalence of a genetic defect that causes leukocyte adhesion deficiency in Holstein cattle

Construction of a DNA probe and detection of Actinobacillus pleuropneumoniae by using polymerase chain reaction

The trigeminal ganglion is a location for equine herpesvirus 1 latency and reactivation in horse

Determination of the detection limit of the polyanerase chain reaction for chicken infectious anemia virus

Differentiation of Trichinella isolates by polymerase chain reaction

Detection of Aspergillus fumigatus by polymerase chain reaction

Random amplified polymorphic DNA analysis of Trypanosoma cruzi strains

Detection of equine arteritis virus following amplification of structural and nonstructural viral genes by reverse transcription-PCR

Detection of Salmonella serovars from clinical samples by enrichment broth cultivation-PCR procedure

Detection of African horse sickness virus by reverse transcription-PCR

Eimeria tenella characterization of a 5S ribosomal RNA repeat unit and its use as a species-specific probe

Direct detection of the porcine reproductive and respiratory syndrome (PRRS) virus by reverse polymerase chain reaction (RT-PCR)

Genotyping bovine kappa casein locus using the polymerase chain reaction (PCR) technique

Relative frequencies of G (VP7) and P (VP4) serotypes determined by polymerase chain reaction assays among Japanese bovine rotaviruses isolated in cell culture

Detection of Theileria sergenti infection in cattle by polymerase chain reaction amplification of parasite-specific DNA

The detection ofAspergillus spp. by the polymerase chain reaction and its evaluation in bronchoalveolar lavage fluid

Detection of chicken anaemia agent DNA sequences by the polymerase chain reaction

Evaluation of the poly3nerase chain reaction (PCR) for detection of Chlamydia psittaci in abortion material from ewes

Detection of canine parvovirus DNA in paraffin-embedded tissues by polymerase chain reaction

Sex determination of bovine embryos by the polymerase chain reaction using y-specific primers

Development of a rapid and sensitive polymerase chain reaction for detection of bovine herpesvirus type 1 in bovine semen

Detection of leptospires in urine by polymerase chain reaction

Sequence analysis of small subunit ribosomal RNA genes and its use for detection and identification of Leishmania parasites

Detection of bovine herpesvirus-1 (BHV-1) genome by PCR

Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples

Rapid detection of bovine herpesvirus 1 (BHV 1) using the polymerase chain reaction

Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis

A rapid diagnostic assay for eastern equine encephalomyelitis viral RNA

PCR-based detection of arboviral RNA from mosquitoes homogenized in detergent

Application of polymorphic DNA sequences to differentiate the origin of decomposed bovine meat

Detection of equine herpesvirus and differentiation of equine herpesvirus type 1 from type 4 by the polymerase chain reaction

Detection of an arbovirus in an invertebrate and a vertebrate host Using polymerase chain reaction

Comparison of two gene amplification methods for the detection of Toxoplasma gondii in experimental infected sheep

Detection of Cryptosporidiumparvum using a specific polymerase chain reaction

Direct polymerase chain reaction detection of Campylobacter jejuni and Campylobacter coli in raw milk and dairy products

16S ribosomal DNA amplification for phylogenetic study

Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction

Detection of Listeria monocytogenes with a nonisotopic polymerase chain reaction-coupled ligase chain reaction

Detection of bovine herpesvirus-1 in bovine semen by a nested PCR assay

Diagnosis and epidemiological association of Listeria monocytogenes strains in two outbreaks of listerial encephalitis in small ruminants

Ligase chain reaction (LCR) -overview and applications

Rapid beta lactoglobulin genotyping of cattle using the polymerase chain reaction

Plasmid based differentiation and detection of Coxiella burnetti in clinical samples

Detection of Coxiella burnetti in cow's milk using the polymerase chain reaction (PCR)

Nucleic acid techniques and the detection of parasitic diseases

Development of a nested-PCR test based on the sequence analysis of epizootic hemorrhagic disease viruses non-structural protein 1 (NS1)

Nested and multiplex polymerase chain reaction for the identification of bluetongue virus infection in the biting midge, Culicoides variipennis

Detection of hog cholera virus and differentiation from other pestiviruses by polymerase chain reaction

PCR detection of ovine herpesvirus-2 DNA in Indonesian ruminants -normal sheep and clinical cases of malignant catarrhal fever

Advances in nucleic acid-based detection methods

Development of a PCR test specific for Leptospira hardjo genotype bovis

Detection of entero and verocyto-toxin genes in Escherichia coli from diarrhoeal disease in animals using the polymerase chain reaction

The polymerase chain reaction: Basic methodology and applications

Laboratory diagnosis of oxacillin resistance in Staphylococcus aureus by a multiplex-polymerase chain reaction assay. Diagnostic Microbiology and Infectious Diseases

Cloning and characterization of ribosomal RNA genes from three species of Haemonchus (Nematoda: Trichstrongyloidae) and identification of PCR primers for rapid differentiation

Detection ofMycoplasma synoviae by polymerase chain reaction

Canine X chromosome-linked hereditary nephritis: A genetic model for human X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the alpha-5 chain of collagen type IV

Diagnosis and molecular epidemiology of the African horse sickness virus by the polymerase chain reaction and restriction patterns

Diagnosis of the African horse siclaless virus serotype 4 by a one-tube, one manipulation RT-PCR reaction from infected organs

Design of onchocerca DNA probes based upon analysis of a repeated sequence family

Detection of Borna disease virus RNA in naturally infected animals by a nested polymerase chain reaction

Polymerase chain reaction for detection of Borrelia coriaceae, putative agent of epizootic bovine abortion

 Sakamoto et al. (1994) ; Stone-Marschat et aL ) Zientara et al. (1993 ) Carrillo et al. (1994 Benkel and Smith (1993) Aly et aL (1993)