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Development of LAMP method for virus detection in white flies

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Development of LAMP method for virus detection in white flies

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Samenvatting

For regional plant protection it is important to detect quarantine plant pathogens or pests that possibly came from transport of products across a border. Therefore, effective, rapid and simple detection methods deployed in the field are required. Q-detect is a European project in which these detection methods are developed. In the molecular detection section several viruses in white flies (Bemisia tabaci) have to be detected[1].
The whitefly can cause damage to many crops by virus transmission. Two of the most common whitefly-transmitted virus genera are viruses from the Begomovirus genus and the Crinivirus genus[2]. The goal of this project was to set up a simple and fast detection method for the detection of six quarantine viruses transmitted by white flies.
In this report the Tomato chlorosis virus(TOCV), Tomato infectious chlorosis virus(TICV) and the Tomato yellow leaf curl virus(TYLCV) are used for the development and optimization of the detection procedure. Based on these results also detection methods for the Cucurbit yellow stunting disorder virus(CYSDV), potato yellow vein virus(PYVV) and Cotton leaf curl virus (CLCuV) will be developed. The detection method is based on a Loop-mediated isothermal Amplification(LAMP). LAMP has some advantages compared to other molecular techniques like PCR. To initiate the LAMP reaction there is no need to denature double stranded DNA into a single stranded form[12]. The amplification reaction takes place continuously under isothermal conditions and amplification can be done with DNA and also with RNA templates. The RNA templates follow the same procedure as with DNA templates, although reverse transcriptase has to be added to the reaction mixture. The amplification efficiency with LAMP is ten times higher than with PCR and is less prone to inhibitors.
By designing 4 primers to recognize 6 distinct regions, the LAMP method is able to specifically amplify the target gene and because LAMP does not require special reagents or sophisticated equipment, the total cost can be reduced [4]. LAMP primersets for the detection of TICV, ToCV and TYLCV in white flies have been developed. Some of the primers could be adapted to make them more stable for all the different isolates, because not all the isolates are completely homologues when compared to each other, this could have an effect on the LAMP reaction. These primers were tested on extracted DNA and/or RNA of the viruses. The primers were also tested on specificity by testing them on closely related viruses. With the use of infected and not infected white flies, LAMP sensitivity was compared with real-time PCR. The primersets have proven to be specific for each individual virus and show the same sensitivity compared to TaqMan assays. The different LAMP primersets can also be combined and be used for universal multiplex screening.
Also different extraction methods were tested for a fast and simple extraction methods applicable for both DNA and RNA. The methods were tested with LAMP and compared with the traditional TaqMan for extraction efficiency. The Quick extract RNA Extraction kit from Epicentre has proven to be fast and easy to perform. It is applicable for ssDNA as well as ssRNA viruses and has an efficient recovery.
In collaboration with the Radboud University of Nijmegen, Institute for Molecules and Materials, we made a start with the development of a micro-fluidic device for LAMP in which amplification and detection can be combined in a single system. Micro-fluidics are devices in which reactions can take place in a relative small volume and are easy to handle.

Toon meer
OrganisatieAvans Hogeschool
AfdelingATGM Academie voor de technologie van Gezondheid en Milieu
AfstudeerorganisatieWageningen UR; Plant Research International; Wageningen
Datum2011-06
TypeBachelor
TaalEngels

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