Molecular Diagnostics

The same principles as used in developing molecular markers can be applied for a range of molecular diagnostic purposes in plants, including:
(i) identification of plant pathogens (viruses, fungi, nematodes,bacteria, insects);
(ii) studying population structure/variations in pathogens;
(iii) identifying the presence and quantifying the presence of transgenes in transgenic foods;
(iv) following possible pollen transfer of transgenes.
All that is needed is to identify specific nucleic acid (RNA or DNA) sequences unique to the target organism and then to develop a reliable extraction/PCR analysis system such that a DNA fragment is only amplified if the target organism or target sequence is present in a sample. The methods and scale by which such analyses (and also marker-assisted selection) can be carried out are advancing rapidly. Analysis can be by:
(i) PCR and gel electrophoresis;
(ii) real-time fluorescent PCR (e.g. using an ABI TaqMan 7700) to quantify the original amount of target sequence without gel electrophoresis;
(iii) matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS);
(iv) use of DNA chips and hybridisation of labelled samples to bound DNA sequences.
The last two methods (MALDI-TOF MS and chip technology), including microarrays ), promise to speed up all DNA fragment analysis applications by an order of magnitude over current gel-based DNA separation technologies.
Typical examples of applications of molecular diagnostics are the following. (1) Routine analysis of farmers’ seed samples (such as lupin) for the presence of seed-borne cucumber mosaic virus (CMV).8 This can done by RT-PCR (to detect viral RNA, sensitivity o1 infected seed per 1000 seeds) or by real-time fluorescent PCR. Farmers must buy clean seed if infection levels of CMV are above 0.5%. (2) Routine analysis of farmers’ lupin seed for the fungal disease anthracnose, caused by Colletotrichum acutatum. A PCR test, based on repeated ribosomal DNA sequences specific to the fungal pathogen, allows infection levels of one seed in 10 000 to be detected. In this case, only clean anthracnose-free seed can be sown.