Plant Virus Detection Assay Development

Contract Number: OMFB-02395/00 (BIO-010/00)

Starting date: 2000. 07. 01.

Duration: 24 months

Total cost: 39,6 M HUF

OM contribution: 19,8 M HUF

Acronym: Plant virus detection

BACKGROUND

The damage that plant viruses can cause is well-known, with their infections they can produce decrease in quantity and quality of crops. The sole protection from such infections is to apply pathogen-free propagation. It is rather important with propagation of wooden stem plants, since the duration of plantations is 10-20 years, by the time the earnings can reach the volume when the expenses can get recovered.

It is a requirement to detect pathogens of plants with the help of PCR in the European Union countries. In Hungary the application of serological diagnostic methods is widespread. Experimental PCR detection is done in few Hungarian laboratories. Detection with PCR is considered to be the most sensitive method with the highest specificity, therefore it is inevitable that the method should come into general use in the Hungarian plant protection and improvement practices.

OBJECTIVES

The aim of the project is to develop a solid phase and enzyme labelled PCR diagnostic assay to detect grapevine leafroll virus RNA. The main advantages of this diagnostic assay are the following: The specific virus RNA separation and purification is very simple and does not take very much time. The enzyme labelled detection system enables to process a lot of samples in a short time.

DESCRIPTION

There are three phases of the development of the virus detection kit.

Phase I: Separation of the leaf roll virus from the plant sample. Biotinylated primer is added to the crude sample as the first step of the PNA template separation. The biotinylated primer is bound to the specific complement sequence on the virus RNA during the hybridisation. In the following step the virus specific template binds on the surface of the GeneFix tube via the biotin-strepavidin bridge. The GeneFix streptavidin tube has been developed by Institute of Isotopes Co., Ltd.

Phase II: The specific RNA template bound the surface of the tube is amplified in a RT-PCR assay. The RT-PCR products are separated from the PCR mixture in a new streptavidin GeneFix tube.

Phase III: The purified RT-PCR product is denatured to ssDNA on the surface of the GeneFix tube. The complement non-biotinylated strand is washed from the tube. The peroxidase (HRPO) labelled probe is hybridised to the biotinylated strand on the tube surface. The enzyme signal shows the presence of the grapevine leafroll virus RNA.

CURRENT SITUATION

At the moment we have finished the first two phases. The virus purification and the main parameters of the RT-PCR have been optimised. Nowadays we are working on to produce HRPO labelled oligonucleotides and developing a chemical conjugation reaction between the enzymes and oligonucleotides.