Occurrence of a variety of symptoms and the emergence of the necrotic PVYNTN has led to a search for more reliable classification tools than simple serological identification.

[8] Previous studies by Visser et al.[9] did not identify any of the local isolates as being PVYC but it has been reported to occur in potatoes in South Africa.

Infection of a potato plant with the PVYO strain results in mild tuber damage and does not cause leaf necrosis.

[17][21] The Agricultural Research Council-Vegetable and Ornamental Plant Institute (ARC-VOPI) 6 of South Africa identified twenty five species of aphid able to function as PVY vectors.

These newly synthesized viral particles are subsequently transported through the plasmodesmata to adjacent plant cells via several assisting potyvirus proteins.

[31] Although the precise mechanism of disease induction by potyviruses in plants is unknown, it is known that these viruses cause a significant shutdown of host gene expression during viral replication.

At the time of symptoms development, research on interaction between susceptible potato cultivar and PVYNTN showed changes in cytokinin level.

[36] In inoculated leaves showing symptoms modifications in chloroplast structure and size,[37] lower chlorophyll levels and differential activity of soluble and ionically bound peroxidases[38] were detected.

[30] The capsid encapsulates a single strand of positive sense RNA which is in the order of 10 kb in length and has a non-translated 5’-terminal region (5’-NTR) as well as a 3’-poly-A tail.

[41][42] The positive sense genome contains a single extended open reading frame and acts directly as mRNA.

Rather than a conventional cap structure, the 5’NTR is associated with a Viral genome linked protein (VPg) which is said to act as an enhancer of transcription.

[28] The 5’-leader sequence has an internal ribosome entry site (IRES) and cap-independent translation regulatory elements (CIREs).

This polyprotein is proteolytically processed by viral proteases (NIa, HC-Pro and P1) and undergoes co- and post-translational cleavage to yield several multi-functional proteins.

Determination of viral status through visual inspection is incredibly difficult as the symptoms may be masked or the infection latent.

Enzyme-linked immunosorbent assay (ELISA) screening of crops and seed potatoes replaced visual inspection in the early 1970s.

The use of ELISA offered routine diagnostic laboratories a quick, effective and sensitive method of screening for a wide range of potato plant viruses.

[45] Solid phases used include nitrocellulose membranes, paper, glass, agarose and polystyrene or polyvinylchloride microtiter plates.

Non-specific binding to the plates is reduced through the use of buffers containing proteins such as casein and non-ionic detergents such as Tween 20.

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.

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.

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.

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.

The most commonly used DNA polymerase is Taq, a thermo-stable enzyme isolated from the thermophilic bacterium, Thermus aquaticus.

Even though RT-PCR is technically more difficult to perform and more expensive than ELISA, it has the ability to allow for the detection of low viral loads.

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 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 offers the researcher the opportunity to amplify and analyze the product in a single tube using fluorescent dyes.