The following points highlight the eight main serological tests which helps in rapid diagnosis of plant viruses. The tests are: 1. Histochemical Test by Immunofluorescence 2. Agar Gel Double Diffusion Test 3. Direct Antigen-coated ELISA 4. Double Antibody Sandwich ELISA 5. Indirect Double Antibody Sandwich ELISA 6. Detection of Virus by Nucleic Acid Spot Hybridisation (NASH) using DNA Probe and Others.
1. Histochemical Test by Immunofluorescence:
This method is suitable to detect viral particles present in protoplasts of plant cells. There are many fluorescent dyes such as rhodamine B (emits red light), Texas red (emits red light), CY3 (emits orange light) and fluorescein (emits green light).
Thin sections of virus-infected seeds/seeds stocks are cut using a microtome. A fluorescent dye is mixed with ү-globulin extracted from the antiserum. Sections containing viral particles are stained with fluorescent stain.
Fluorescent dye-tagged immunoglobulin enters into the infected tissues. The antigen-antibody reaction takes place. When specimen is exposed to UV light, the cells emit fluorescence of varying colours (based on type of dyes) inside which immunoglobulin-tagged dye has bound with antigen. This gives a positive result. The fluorescence can be detected through a fluorescent filter fitted in a fluorescent microscope.
2. Agar Gel Double Diffusion Test:
This test is performed to determine the antigenic relationship of the viruses. Petri plates containing gelled agar plates are used to prepare wells. Wells are prepared in such a way that one well must be in centre and the others to its periphery.
The peripheral wells are filled with purified viral suspension and central well with similar amount of known antiserum. If antigens react positively with any antiserum, fusion of precipitin line (spur) is observed. This shows that the two isolated strains are identical. Since both the reactants diffuse into the well, it is called double diffusion test.
3. Direct Antigen-Coated ELISA (DAC-ELISA):
This technique was developed by Hobbs (1987). In this method an assay is performed on a polystyrene microtitre plate. The crude antigen is prepared by using fresh leaf tissues of infected parts of the plant in coating buffer and extract is prepared through maceration of tissues. Pure extract is obtained after centrifugation. The extract is placed into wells and incubated at 37 °C for 1 hour. Then antigens are washed.
The plates are blocked with milk powder solution for 1 hour. Polyclonal rabbit antibody and anti-rabbit immunoglobulin alkaline phosphatase conjugates are used at 1:1000 and 1:30,000 dilution, respectively. The reaction is read at 405 nm after 1 hour of adding substrate (p-nitro-phenyl phosphate) by an ELISA- reader.
4. Double Antibody Sandwich ELISA (DAS-ELISA):
This technique was developed by Permar (1990). DAS-ELISA techniques has been used for detection and identification of strains of viruses. Polyclonal antiserum (PAb) detects the presence of virus up to 10-3 dilution. Differentiation of mild and severe strains is done using monoclonal antibodies (MAb). Strain differentiation achieved by MAb depends on its structural epitope.
Small amount of infected plant tissues is transferred in phosphate buffer and macerated. Wells of ELISA plate are coated with y-immunoglobulin (IgG). Plate is incubated for 1-4 hours and washed with water.
Enzyme-tagged with IgG diluted in conjugated buffer is added to each well and incubated for 1-4 hours. It is washed and substrate solution is added. After 15 minutes optical density is measured at 405 mn in an ELISA reader.
5. Indirect Double Antibody Sandwich ELISA (I-DAS-ELISA):
Nikolaeva (1998) found that MAb (e.g. MCA-13) cannot distinguish between two isolates among the severe strains of virus. To overcome this problem, they developed a set of primary/secondary combination of trapping and detecting antibodies in an I-DAS-ELISA.
Nikolaeva put forth the information on epitope-specificity of a range of viral specific antibodies which helped to develop a serological system to distinguish between viral isolates. In this techniques a polyclonal antibody is used for coating the plate and a secondary antibody R-109 was used before adding the conjugate.
The tissues of test sample are macerated and left for 6 days at 4°C. The sample is processed as described for DAS-ELISA. A dilution series of two antibodies is run to standardize the dilution combination CH110/R 109. Based on optimum combination dilution for testing strain was chosen as 1:1,00,000/ 1:15,000. The other details are as described for DAS- ELISA. Early colour development is considered to be non-specific.
6. Detection of Virus by Nucleic Acid Spot Hybridisation (NASH) using DNA Probe:
This technique is used for detection of viral DNA in symptom-less host. Small samples (e.g. leaves, seeds, etc.) are collected from each plant after 21 days of inoculation. Total DNA from these samples is extracted and stored at -70°C for NASH. Prepared DNA sample is heat-denatured to get a concentration of 2.5 and 0.25 microgram of total DNA. DNA is dotted on nitrocellulose membrane. The membrane is dried and baked at 80°C for 2 hours in vacuum.
Virus-specific DNA probe is prepared from the clones of virus present in a plasmid (e.g. pUC18) after incorporating (α-32p)-dCTP by random priming method. The probes of different size are generated using restriction enzymes that separates the viral DNA insert from the plasmid. Further restriction of the viral DNA by different restriction enzymes yields the viral DNA fragments of different size. These fragments are tagged with (a-32p)-dCTP.
The radio-labelled probes are added to pre-hybridisation solution. The baked nitrocellulose membrane is transferred in pre-hybridisation solution. Pre-hybridisation and hybridisation are done at 65°C for 4 hours and 18 hours, respectively.
The hybridised blots are washed in 2X saline buffer thrice for 15 minutes at 65°C. The membrane is exposed to X-ray film for 24-48 hours at -70°C and auto-radiographs are prepared. The hybridised viral DNA will form blots and indicates the presence of suspected virus in the sample.
7. Electron Microscopy:
Electron microscopy helped in identifying the presence of viruses in plant tissues. For example, TMV studies were done by Stanley (1935). TEM studies are made in the leaf dip preparation which were infected by potyviruses.
TEM studies revealed the presence of flexuous rods (750×12 nm). Ultra-thin sections of infected chilli leaves revealed the presence of cytoplasmic inclusion called as ‘pinwheels’ inclusion bodies which are typical of potyvirus group.
8. Dot Immuno Binding Assay (DIBA) or Dot ELISA:
In this method nitrocellulose membrane is used as solid substrate for ELISA. Because nitrocellulose membrane can easily be transported to detect a large number of samples in field. Using small quantities of antiserum virus present even in small quantities can be detected.
Plant extract from infected tissues is prepared and blotted onto nitrocellulose membrane or enzyme-linked ү-immunoglobulin is coated onto it. A substrate is added to develop final colour. The substrate (nitro-blue tetraxolium, 5-bromo-4-chloro- 3-iodylphosphatep-toludine salt and formamide) is hydrolysed and produces bright stain which can be observed by eyes or a reflectance densitometer.