Some Selected Serological Diagnostic Techniques in Plant Virology

Particle morphology, host range and the serological properties of the coat protein are generally being used to identify plant viruses. In classification and the establishment of taxonomic relationships, cross-reactivity of antisera raised against viruses from different groups have been frequently used (KPL Technical Guide on line 1999/2000 edition). However sequence data of nucleic acid are accumulating rapidly and are allowing more accurate relationships to be established between the individual members of virus groups than serological methods do (Dellaporta et al. 1983; Jackie Hughes et al 2004). Sequencing parts of a virus genome is often done in the identification of a virus if detailed serological analysis cannot provide conclusive data about the virus (Dellaporta et al. 1983). This involves purification and isolation of the virus particles and the subsequent cloning of parts of the virus. Using advance molecular methods in specific Polymerase chain reaction (PCR), new virus sequence data can be obtained without the need to purify a virus or to clone parts of its genome (Jackie Hughes et al 2004). Advances in molecular biotechnology have contributed immensely in developing specific and sensitive diagnostic assays and sensitive antisera against individual viruses or group of viruses (Dellaporta et al. 1983; Jackie Hughes et al 2004). Useful tools are provided in diagnostic procedures for virus disease monitoring.


Introduction
Particle morphology, host range and the serological properties of the coat protein are generally being used to identify plant viruses. In classification and the establishment of taxonomic relationships, cross-reactivity of antisera raised against viruses from different groups have been frequently used (KPL Technical Guide on line 1999/2000 edition). However sequence data of nucleic acid are accumulating rapidly and are allowing more accurate relationships to be established between the individual members of virus groups than serological methods do ; Jackie . Sequencing parts of a virus genome is often done in the identification of a virus if detailed serological analysis cannot provide conclusive data about the virus . This involves purification and isolation of the virus particles and the subsequent cloning of parts of the virus. Using advance molecular methods in specific Polymerase chain reaction (PCR), new virus sequence data can be obtained without the need to purify a virus or to clone parts of its genome (Jackie . Advances in molecular biotechnology have contributed immensely in developing specific and sensitive diagnostic assays and sensitive antisera against individual viruses or group of viruses Jackie Hughes et al 2004). Useful tools are provided in diagnostic procedures for virus disease monitoring.

Virus diagnosis
Different diagnostic techniques are used in the identification of plant viruses. Symptom observations is an important tool in field virus diagnosis though it is not a conclusive methods, it is combined with other methods (Fajinmi, 2006

Types of serological tests
There are principally two types of serological test, the liquid phase test and the solid phase test (Jackie . Both methods are basically interaction between known antibodies and the unknown virus for positive identification, but visualization differs. In liquid phase test, there is formation of a visible precipitate in a solution reaction, i.e. agglutination of visible cells. This kind of test include, gel diffusion test, precipitin or microprecipitin test (Jackie . In solid phase test, it is an enzymatic reaction, where antibodies are conjugated with a marker enzyme in which the antibody attaches itself with the antigen after recognizing it (Jackie . The chosen enzyme reacts with the substrate to provide a positive identification. This is confirmed through colour change in the substrate. Examples of this test include micro-titre plates or nitrocellulose membranes (Jackie Hughes et al 2004).

Test methods selection
Test method could be selected depending on the reason of the serological analysis, the information required and availability of materials to be used (Jackie . Diagnosis is performed in optimum conditions with proper controls and must be reproducible. This will enhance proper, correct and desirable data analysis for the result to be valid. Availability of laboratory equipment and antibodies will determine the specific serological test to be used (Jackie Hughes et al 2004).

Double diffusion tests
The materials needed to design, perform and evaluate double diffusion tests includes, (1) Pipette (10 µl -100 µl is preferred; other pipettes can be used) (2) Flat bottomed glass or plastic dishes (Petri dishes are recommended) (3) Agar (4) phosphate Buffer (5) cork borer for punching out well patterns (6) tight sealed moist chamber for incubation with moist towel in bottom (Jackie Hughes et al 2004).
Test Procedure as described by Jackie  a. Dissolve 70mg of agar in 100mls of buffer (0.7 % W/V) and autoclave at 120 O C for 10 minutes, Cool to 60 O C. b. Dispense to sterilized Petri-dishes and allow to fully solidify. c. With the aid of a cork borer, punch out well patterns on the solidified agar in the Petridish with no damage to the clean -cut sides of the wells. d. Dispense the antibodies and antigen to the wells according to the test pattern. e. Incubate text plate in the moist chamber for 5-7 days.

Interpreting double diffusion test results
There is a radial diffusion of the antigen and antibodies form the wells. Lines of precipitate are formed in the agar as the antibodies and antigen meet; results are interpreted based on these patterns of precipitate (Jackie Hughes et al 2004). Combination of antibodies and antigen must be properly balanced with the correct dilutions for correct interpretation (Jackie Hughes et al 2004).

Enzyme-Linked Immunosorbent Assay (ELISA) tests
This is the most common technique for diagnosing of plant viruses and it is a solid phase assay. The test reaction involves antibody and antigen reaction to produce a positive reaction. The reaction takes place in a microtitre plates made of either polystyrene or polyvinyl chloride (PVC) (KPL Technical Guide (on line 1999/2000 edition); Jackie . Antibodies conjugated to a "marker enzyme" are used in ELISA. Reaction between the antibody and the antigen will make the "marker enzyme" to react in the substrate resulting in colour development (KPL Technical Guide on line 1999/2000 edition); Jackie Hughes et al 2004).
There is "direct" and "indirect" ELISA test. In "direct" ELISA test, the detecting antibody bounds with the marker enzyme while in "indirect" ELISA test, the antibody enzyme conjugate binds with the detecting primary antibody and not directly with the virus (KPL Technical Guide on line 1999/2000 edition); Jackie ).
"Direct" ELISA procedure as described by KPL technical guide (on line 1999/2000 edition) and Jackie  Coat the microtitre plate with antibodies followed by the virus sample. The virus becomes bound to the antibodies for a positive reaction. Wash the plates with phosphate buffer in Tween-20 to remove any of the virus samples that has not reacted with the antibodies. Add antibody enzyme conjugates then wash again to remove excess unbound conjugate then add the substrate. Colour development provides and indication that the virus and the antibody have reacted. This shows that the antibody enzyme conjugates has attached to the trapped virus allowing the enzyme to r e a c t . T h i s c o n f i r m s a t e n t a t i v e v i r u s identification and quantification. This test method is referred to as double antibody sand which (DAS) ELISA because the virus is sandwiched between the capturing antibody and the detecting antibody. This method is strain specific and the antibody to detect the virus must be conjugated to an enzyme and a specific conjugate is required for each antibody to be used.

"Indirect" ELISA
This involves the usage of antibodies which have been raised to two different animals and using different methods for capturing the virus (KPL Technical Guide on line 1999/2000 www.intechopen.com edition). Though the antibodies that identify with the virus have been raised for that specific virus, the secondary antibody marker can be raised to recognize a wide range of primary antibodies, with the fact that the primary antibodies were produced in the same animal species against which the secondary antibodies were raised (Jackie . It is cheaper to produce a secondary marker antibody enzyme conjugate in "indirect" ELISA than primary marker antibody enzyme conjugate in "direct" ELISA (Jackie . Two different animals are used in the production of primary and secondary antibodies (Jackie . In recognizing specific viruses, primary antibodies are raised in one animal species, while production of secondary antibodies is to recognize proteins from specific species of animal ( Jackie Hughes et al 2004). For instance, antibodies as protein source from rabbit can be used as antigen to produce secondary antibody from mouse ( Jackie Hughes et al 2004). These secondary antibodies from mouse can bund with any antibodies produced in a rabbit.
Examples of "indirect" ELISA methods as described by KPL Technical Guide (on line 1999/2000 edition) and Jackie  include; Antigen coated plate (ACP) or plate trapped (PTA), direct antigen coated (DAC) assays. In this assays, the first to be added is the primary antibodies, then the secondary antibody enzyme conjugate and substrate. In triple antibody sandwich (TAS) ELISA, three antibodies are used in a sequential order. Trapping antibodies followed by the virus sample then the primary antibodies added followed by the secondary enzyme conjugated antibodies which binds with the primary antibodies. Protein A sandwich (PAS) ELISA uses protein A for an increase in the test specific and reaction sensitivity by controlling orientation of antibodies. Steps involve; Plate covered with protein A  trapping antibodies virus sample secondary antibody. Protein A will detect by biding with the secondary antibodies if in correct orientation.

ELISA result interpretation
There are two basic type of ELISA reader. One reads the entire plate at once while the other reads individual well if correctly used. Plate's bottom to be read must be clean and dry and result read at room temperature. Contact the user's manual for the ELISA reader for further www.intechopen.com assistance. ELISA result is read using nanometer reading set at A405 infection (A 405 inf ) no matter the type of ELISA reader used (Jackie . A sample test is considered positive for the virus if its ELISA reading doubles that of the negative control (KPL Technical Guide on line 1999/2000 edition). In some cases the sample test is considered virus positive if the ELISA reading is1.5 times the means of the negative controls (Jackie Hughes et al (2004);

Polymerase Chain Reaction (PCR)
This is a method for nucleic acid amplification in-vitro. The product of the polymerase chain reaction is been used by PCR for identification. It is highly sensitive; apart from identifying the DNA it also amplifies the target nucleic acid sequence . It uses multiple cycles of template denaturation, primer annealing and primer elongation to amplify DNA sequence ; Jackie Hughes et al 2004). Each step occurring at specific temperature for specified time period.
Components of PCR as described by Dellaporta et al. (1983) and Jackie  Template: Nucleic acid of infected plant or virus DNA. RNA viruses are converted to cDNA before amplification.
Primers: Sequences of short fragment of oligonucleotides (single stranded DNA) complementary to the sequences at the end of target sequences to be amplified. Forward and reverse primers are required in this reaction. dNTPs: Nucleotides required for the extension of the newly synthesized DNA strand. dATP, dCTP, dGTP, dTTP (the four neucleotides) are included in the mixture Enzyme: A thermostable (heat resistant) enzyme is required for polymerization due that the polymerase reaction undergoes denaturation of template at high temperature. Taq polymerase is the thermostable polymerase enzyme used frequently.

Reaction buffer and magnesium chloride
The magnesium ion concentration affects among others, enzyme activity, primer annealing and denaturation (Jackie . PCR reaction is carried out in a thermo cycler that has been programmed to cycle between high and low temperatures within a specified period of time. This involves 3 main steps as described by Jackie  Two new DNA strands as explained by Jackie  are formed at the end of the first cycle totaling four strands of DNA template which serves as templates for the second cycle. In the second cycle, the new DNA elongates as the primers binds to the templates. A total of eight strands would have been synthesized at the end of the second cycle. The DNA increases at exponential rate with each cycle as the PCR continues until set number of cycles has been reached. Ethidium bromide stained gel is used to visualize the final product Jackie Hughes et al 2004). PCR Reaction mixture as described by Dellaporta et al. (1983) and Jackie  To detect individual viruses, specific primers are to be used.

Reagent
Stock

Reverse Transcriptase PCR (RT-PCR)
Viruses with RNA are amplified with reverse transcriptase as their nucleic acid in PCR. DNA is used as template for Taq polymerase; therefore the virus will have to convert its RNA to cDNA before the polymerase reaction. Short C DNA are formed due to cleavages caused in the RNA due to the contaminating RNase. One-tube reaction or two-tube reactions can be used to carry out RT-PCR ; Jackie Hughes et al 2004).

Two-tube RT-PCR
RNA extracted from the virus as template or total nucleic acid extracted from the infected plant is used to synthesize C DNA by reverse transcription. An aliquot of the reverse transcription reaction is used in the PCR. Usually, not more than 1/5 of the total PCR mixture should derived from the reverse transcription reaction (Jackie Hughes et al 2004).

One-tube RT-PCR
This is where the same buffer is used to carry out the reverse-transcription reaction and PCR so the two reactions can not be separately optimized. Before adding the mixture, the RNA need to be denatured at 70-75 O C for 5 min so that there can be good synthesis of C DNA which is immediately followed with a PCR cycling programme ( Jackie Hughes et al 2004).
The thermocycler can accommodate both programmes at the same time as one programme (Jackie Hughes et al 2004).

Immunocapture PCR
Along with the PCR the virus particle could be trapped using the antiserums. This reaction could be carried out in a single tube. This method is good for viruses having low concentration in the plant or viruses having their genome integrated into host plant genome ; Jackie Hughes et al 2004).

Immunocapture reverse transcription PCR
In this test RNA is used as the template which makes it differs from immuno-capture PCR which make it very efficient in detecting RNA in viruses from plant that could have affected the enzymes in the reverse transcription reaction or the PCR through some inhibitory compounds that it contains ( Jackie Hughes et al 2004). A single tube can be used to carry out immuno-capture, reverse transcription and the PCR and the different reactions could be carried out separately.
An example of a typical IC-RT-PCR as described by Jackie

Extraction of DNA for PCR
The DNA extraction procedure as developed by Jackie .
13. Decant the ethanol and air-dry the DNA (at room temp) until no trace of alcohol can be seen in ht tube.
The DNA is re-suspended in 950 µl TE or sterile distilled water) and stored in the freeze as stock solution. The DNA is usually diluted 1x10 5 times for PCR, but it will be better do a dilution curve to determine the best dilution for amplification.
Extraction buffer TE buffer 100 mM (pH 8.0) 10 mM Tris 8.5 mM EDTA 1 mM EDTA 500 mM NaCl 10 mM β-mercaptoethanol (added just before use) Extraction of total nucleic acids for RT-PCR (Method A) as described by Jackie Hughes et al (2004)