ROLE OF MOLECULAR TECHNIQUES IN DIAGNOSIS OF ANIMAL DISEASES
DR RAJEEV KUMAR, SCIENTIST , ICAR
Laboratory diagnostics for Veterinary pathogens have traditionally relied on methods of detecting the pathogen by culture or antibodies, using a variety of techniques such as neutralisation, enzyme-linked immunosorbent assay, agar gel immunodiffusion and complement fixation. The effective control and treatment of diseases of animals requires access to diagnostic tests that are rapid, reliable and highly sensitive. However, these methods are time-consuming and costly.
Efforts to overcome these problems have lead to the development of several
diagnostic methods including fluorescent antibody tests (FAT), enzyme-linked
immunosorbent assays (ELISA), radioimmunoassay (RIA), in situ hybridization (ISH), dot blot hybridization DBH) and polymerase chain reaction (PCR) amplification techniques. The use of DNA-based methods derives from the premise that each species of pathogen carries unique DNA or RNA sequences that differentiate it from other organisms. The techniques offer high sensitivity and specificity, and diagnostics kits allowing rapid screening for the presence of pathogen DNA are moving rapidly from development in specialized laboratories to routine application. DNA probes are expected to find increasing use in routine disease monitoring and treatment programs in veterinary medicine, in field epidemiology and in efforts to prevent the international spread of pathogens. The development and use of DNA-based diagnostic techniques will assist international efforts to control the introduction of exotic diseases into new geographic areas. Reliable and rapid techniques are needed by national and regional diagnostic laboratories to screen for important pathogens.
The use of nucleic acid-based diagnostics in veterinary medicine has increased
exponentially in recent years. These techniques have redefined the level of information
available for animal disease control programmes.In addition, modifications of nucleic acid
detection techniques based on polymerase chain reaction (PCR) have lead to the development of rapid, specific assays. The molecular technique with the widest variety and application in veterinary diagnostics is PCR. The strength of this technique is its ability to make millions of copies of a deoxyribonucleic acid (DNA) target. This amplification enables the desired target to be readily detected by other techniques such as electrophoresis and sequencing. Initial use of PCR in veterinary diagnostics was for specific genomic detection e.g. bovine viral diarrhoea virus, foot and mouth disease virus, Infectious bovine rhinotracheitis, Buffalopox, Ephemeral Fever virus, etc.
Following are the list of the different molecular diagnosis methods in animal disease:
1. Reverse transcription PCR
2. Nested PCR
3. Real-time PCR
4. Multiplex PCR
5. Fluorescent in situ hybridisation (FISH)
6. Nucleic acid sequence-based amplification (NASBA)
7. Proteomics
8. Microarray technology
9. Nanotechnology
Reverse transcription PCR: ——–
This assay initially makes a complementary DNA copy of the original viral RNA before final amplification and is more sensitive than the traditional Northern blot method of RNA detection. A potential approach to RNA detection is to use binding stretches of RNA (aptamers). This type of application does not require amplification and is currently used in studies of gene expression in human cells and tissues. Most of the disease causing RNA viruses are diagnosed now a days using reverse transcriptase PCR converting their fragile RNA genome into stable cDNA.
Nested PCR: ———
It refers to the application of a second set of primers targeting a shorter area on the first-stage amplified product (DNA). Using this approach increases the sensitivity of the PCR and generates two amplified products for confirmation purposes. This technique has been used to detect a number of agents of veterinary interest including West Nile virus (1). A disadvantage of the nested PCR is the increased risk of cross-contamination due to the opening of amplification tubes to add an additional set of primers.
Real-time PCR is the latest improvement in the standard PCR technique to be implemented in veterinary laboratories. This technique is a single-capillary, closed assay that greatly decreases the problem of cross-contamination between samples. The fluorescence readings are plotted by computer software and results can be transmitted electronically, eliminating the need for post-PCR reaction analysis by electrophoresis (2). The development of extraction methods such as the magnetic bead technique has made it possible to use real-time PCR to test large numbers of samples in a matter of hours during disease outbreaks. In addition, realtime PCR has been adapted for use in the field through the use of portable thermocyclers and lyophilised reagents. This approach may allow for more rapid decision-making during potential disease outbreaks. The PCR is also used extensively for the genotyping and phylogenetic analysis (relatedness) of veterinary pathogens.
Non-PCR methods of nucleic acid detection: ——
New methods of nucleic acid amplification have been developed and may eventually
be used for veterinary diagnostics. Examples of these methods include the rolling circle
amplification technique and direct signal amplification systems. These techniques are
currently being used in human diagnostics for the detection of human cytomegalovirus and
human immunodeficiency viruses;veterinary applications are currently being developed.
Fluorescent in situ hybridisation (FISH) is a technique that can localize nucleic acid
sequences within cellular material. Peptide nucleic acids are molecules in which the sugar
backbone has been replaced by a peptide backbone. These molecules are perfect mimics of DNA with high affinity for hybridisation that can be used to improve FISH techniques.
Nucleic acid sequence-based amplification (NASBA) is a promising gene amplification method. This isothermal technique is comprised of a two-step process whereby there is an initial enzymatic amplification of the nucleic acid targets followed by detection of the generated amplicons. The entire NASBA process is conducted at a single temperature, thereby eliminating the need for a thermocycler.
Proteomics ——–
In addition to the use of proteomics to identify and characterize the protein produced
by pathogenic agents, proteomic technologies have great potential in veterinary diagnostic
applications because they target the patterns of protein expression of the target analyte
whether it is viral, bacterial, parasitic, etc. The standard proteomic approach involves the
separation of proteins by two-dimensional gels with the staining of the proteins and
molecular weight control. This protein ‗pattern‘ or fingerprint is then analyzed by performing image analysis . Proteome maps can be compared in order to find proteins that may be up- or down-regulated due to disease. A protein of interest can be cut from the gel and fully characterized using peptide-mass fingerprinting and/or mass spectrometry methods. In the future, veterinary diagnostics may make use of proteomics to identify or look for known disease markers or patterns with biochip technology and instrumentation that combines mass spectrometry with other separation chromatography or molecular techniques. These instrumentations are designed to specifically select, separate by molecular mass, and identify the complex mixture of proteins in a sample, which can then be compared to known samples for diagnostic purposes. . This type of technology may be useful for identifying animals infected with agents that do not induce predictable serologic reactions, such as bovine tuberculosis .
Microarray technology ———
Originally developed for the mapping of genes, it is being used to detect a wide
variety of veterinary pathogens .Specific oligonucleotides are bound to small solid
supports such as glass slides, silicon chips or nylon membranes.Extracted DNA or
complementary DNA is labelled with a fluorescent dye and then hybridized with the
microarray. Specific patterns of fluorescence are detected by a microarray reader which
allows the identification of specific gene sequences found only in the veterinary pathogen of
interest. This technology has the potential to identify the presence of agents of interest at the serotype or subspecies level, or to differentiate agents that cause similar clinical signs, for example, vesicular lesions.
Nanotechnology ——–
The term nanotechnology‘ is broadly defined as systems or devices related to the
features of nanometre scale (one billionth of a metre). This scale of technology as it applies to diagnostics would include the detection of molecular interactions. The small dimensions of this technology have led to the use of nanoarrays and nanochips as test platforms . One advantage of this technology is the potential to analyze a sample for an array of infectious agents on a single chip. Applications include the identification of specific strains or serotypes of disease agents, such as the identification of specific influenza strains, or the differentiation of diseases caused by different viruses but with similar clinical signs, such as vesicular viral diseases. Many research groups are considering the use of chip assays that detect a number of agroterrorism agents in each sample. Small, portable platforms are being designed to allow pen-side testing of animals for diseases of concern. Another facet of nanotechnology is the use of nanoparticles to label antibodies. These labelled antibodies can then be used in various assays to identify specific pathogens, molecules or structures. Examples of nanoparticle technology include the use of gold nanoparticles, nanobarcodes, quantum dots (cadmium selenide) and nanoparticle probes.
Impediments to the Use of DNA-Based Diagnostic Techniques —-
Although offering considerable potential, the routine use of DNA-based diagnostic techniques is hampered by a number of potential problems.
• The extreme sensitivity of these methods allows the detection of target DNA present
at very low levels. However, positive results provide little quantitative assessment of
the infection level, and do not indicate whether the pathogen is replicating or causing
disease in the species tested. Thus, carrier status and viability of the pathogen are not
determined using DNA-probes.
• The extremely high specificity of these tests, coupled with the ability of many viruses
to rapidly change in genetic structure, can result in failure to detect a virus that has
altered its genetic profile.
• Large differences in sensitivity are related to the PCR method used
• PCR methodologies are highly susceptible to contamination. Contamination during
processing may result in false positives, particularly in 2-step PCR methods. PCR
tests must be conducted in very well managed, clean laboratories.
• “False negatives” are easily caused by the selection of inappropriate host tissue
sources for detection of the pathogen in question, incorrect choice of DNA extraction
method, or low pathogen prevalence in the population sampled.
DNA-based detection and diagnostic methods have the potential for widespread
application in Animal diseases diagnosis . As the technology is already being adopted
rapidly in developing countries in Asia, there is an urgent need to address these issues and to develop an action plan for research and training activities that will facilitate more effective utilization.
