ELISA In the past, crops were inspected visually to determine whether or not they were disease free. Visual inspection was also used as a basis for seed certification. Determination of viral status through visual inspection is incredibly difficult as the symptoms may be masked or the infection latent. The efficiency to which the antigen binds to the solid phase is dependent on temperature, length of exposure as well as concentration. Nonspecifically bound components are less strongly bound than the specific bound ones. Detection is achieved either through the addition of an enzyme-coupled antibody or the addition and detection of a biotinylated antibody. In a system using an enzyme-coupled antibody the subsequent addition of an appropriate substrate results in the formation of a colour proportional to the amount of antigen. This may be overcome through the use of a biotin-avidin or biotin-streptavidin bridge. In this type of system
biotin is coupled to the antibody. The biotin molecule has no influence on the working of the antibodies and is easily detectedusing avidin or streptavidin conjugated to a suitable enzyme. Streptavidin has an extremely high affinity for biotin which results in even a higher degree of specificity than a system in which the enzyme is coupled directly the antigen. To establish whether or not the antigen is present, a substrate specific for the enzyme used is added. The enzyme then converts the substrate to a coloured product and the colour intensity can be correlated to the amount of antibodies bound and thus the amount of antigen present. A DAS-ELISA has the advantage that it can increase the specificity of the ELISA and reduce the occurrence of non-specific binding. As a result, the DAS-ELISA principle is commonly employed in ELISA's for the detection of plant pathogens in plant sap without prior purification of the pathogen. The ELISA is considered to be a safe, inexpensive and rapid method for detection of plant viruses. The inexpensive nature and relative simplicity thereof allows for it to be used as a workhorse within the agricultural sector and is used to screen thousands of samples per year. Unfortunately ELISAs are not completely failsafe. Virus levels within potato tubers, which are screened by ELISA for use as seed potatoes, are normally low while the tubers are dormant. ELISA detection of viruses in these potatoes is difficult and absorbance values may fall below the set cut-off value. For this reason, seed tuber screening is performed on sprouting rather than dormant tubers. Although this results in more reliable readings than direct tuber testing, it does delay the certification of seed potatoes. Another disadvantage of an immuno-based detection method is that changes at the gene level may have an influence on the immunogenicity of the antigen to be detected. In terms of potato plant viruses, mutations within the CP gene may cause the CP to undergo conformational changes rendering antibodies produced against the previously present virus less effective.
RT-PCR Reverse transcriptase polymerase chain reaction (RT-PCR) has become a powerful and effective method for detection of potato plant viruses within potato plant material and even dormant potatoes. Only a minute piece of plant material is required for analysis using RT-PCR. Considering the protocol described within this thesis, 0.1 g of plant material is enough for 14 500 separate reactions. During a RT-PCR specific target RNA sequences are amplified exponentially into DNA copies. For this to occur, however, the RNA of the virus must first be transcribed to DNA by means of a reverse transcriptase polymerase. This polymerase synthesizes a DNA strand using the RNA as template. This results in a DNA/RNA complex. For synthesis of a DNA strand from the RNA template only the reverse primer is required since the RNA is a single strand arranged from 5’ to 3’. Subsequently, the newly synthesized DNA strand is used as a template for traditional PCR. Different types of reverse transcriptase polymerases are available to suit different needs and reaction conditions. Reverse transcriptase enzymes commonly used include AMV RT, SuperScript III, ImProm-II, Omniscript, Sensiscript and Tth RT. At the end of the RT step the polymerase enzyme is heatactivated. It could also be that the reverse transcriptase polymerase and DNA polymerase is one and the same enzyme and that the enzyme only requires a DNA polymerase activation step after the RT step. An example of such an enzyme is Tth polymerase. This enzyme has both RNA-dependent reverse transcriptase and DNA-dependent polymerase activity. However, the active center of the DNA polymerase is covered by dedicated
oligonucleotides, called
aptamers. At temperatures below the optimal reaction temperature of the DNA-dependent polymerase component of Tth remains covered by the aptamers. At these temperatures the Tth enzyme only synthesizes a DNA copy of the RNA template. Once the reaction temperature is raised to 95 °C, the aptamers are removed and the DNA-dependent polymerase component will start to amplify the target sequence. PCR amplification of the DNA target occurs in three steps:
denaturation,
annealing and extension. RT-PCR also allows for the detection of several viral targets in the same reaction through the use of several primer combinations. This is called multiplexing. Although multiplexing is technically more demanding than a traditional simplex reaction it allows for a higher throughput in that a single sample can be tested for several viral strains in a single reaction. Primers used for multiplexing are chosen in such a manner that they result in amplicons of various sizes. This allows for post RT-PCR analysis using gel electrophoresis. Although RT-PCR saves time, allows for multiplexing and is more sensitive than ELISA, the reagents and instrumentation needed are expensive and require a higher level of technical expertise. Also, end product analysis using gel electrophoresis is laborious, relatively more expensive, time-consuming and does not lend itself to automation. For these reasons the use of RT-PCR for routine screening is not feasible and has not replaced ELISA. It does, however, provide the industry with the opportunity to screen borderline cases, especially in the case of seed potato certification.
Quantitative PCR In most traditional PCRs the resulting products are analyzed after the PCR has been completed. This is called end-point analysis and is normally qualitative of nature rather than being quantitative. For this sort of analysis, products are mostly analyzed on an
agarose gel and visualized using
ethidium bromide as a
fluorescent dye. Direct correlation between signal strength and initial sample concentration is not possible using end-point analysis since PCR efficiency decreases as the reaction nears the plateau phase.
Quantitative PCR, however, offers an accurate and rapid alternative to traditional PCR. Quantitative PCR offers the researcher the opportunity to amplify and analyze the product in a single tube using fluorescent dyes. This is known as homogeneous PCR. During a quantitative PCR the increase in fluorescence is correlated with the increase in product. Through the use of different specific, dyes quantitative PCR can be used to distinguish between different strains of a virus and even to detect point mutations. The major advantage of quantitative PCR is that analysis of resulting products using gel electrophoresis is not required. This means that quantitative PCR can be implemented as a high-throughput technique for sample screening. Quantitative PCR has been described for detection and discrimination of PVYO and PVYN isolates and for reliable discrimination between PVYNTN and PVYN isolates. == See also ==