Print-capture PCR for detection of tomato begomoviruses from plants and whiteflies

Abstract

Print-capture (PC) Polymerase chain reaction (PCR) was evaluated as a novel detection method of plant viruses. Tomato (Lycopersicon esculentum) plants infected with begomovirus (fam. Geminiviridae, gen. Begomovirus) and viruliferous whiteflies were used to study the efficiency of the method. Print-capturing steps were carried out using non-charged nylon membrane or filter paper as the solid support for DNA printings. Amplified DNA fragments of expected size were consistently obtained by PCR from infected plants grown in a greenhouse, after direct application of printed materials to the PCR mix. However, virus detection from a single whitefly and from field-grown tomato samples required a high temperature treatment of printed material prior to PCR amplification. Comparison of nylon membrane and filter paper as the solid support revealed the higher efficiency of the nylon membrane. The application of printcapture PCR reduces the chances of false-positive amplification by reducing manipulation steps during preparation of the target DNA. This method maintains all the advantages of PCR diagnosis, such as the high sensitivity and no requirement of radioactive reagents.

Comparative research of effectiveness of cellulose and fiberglass porous membrane carriers for bio sampling in veterinary and food industry monitoring

“…Late diagnostics of virus infections results in livestock deaths, crop decrease and general financial losses. The technique described in this paper is effective for sample storage with further foreign DNA detection by means of PCR analysis in plant [32][33][34] and animal [19,21,[35][36] biosamples. Due to active development of genetic engineering a demand arose in agriculture and food industry for control over quantity of genetically modified products.…”

Detecção de um begomovírus em amostras foliares de tomateiro com sondas não-radioativas

“…A transmissão desse vírus se dá pela moscabranca, Bemisia argentifolii Bellows & Perring (SANTOS et al, 2004). A diagnose é, em geral, realizada pela avaliação dos sintomas e por meio de testes de detecção por "polymerase chain reaction" (PCR) e por hibridização com sonda radioativa (SANTANA et al, 2001;NAGATA et al, 2004). Os métodos de hibridização com sonda não-radioativa já vêm sendo utilizados para detecção de begomovírus fora do Brasil (SÁNCHEZ-CAMPOS et al, 1999).…”

“…A síntese da sonda foi feita utilizando-se o DIG-High prime labeling kit (Roche) seguindo-se as recomendações do fabricante, usando 3μg do DNA amplificado do clone pCP por PCR com os oligonucleotídeos CP1 e CP2 (fragmento de 0,9kb), contendo todo o gene da capa protéica (NAGATA et al, 2004).…”

Bidens Mosaic Virus is a Member of the Potato Virus Y Species

“…This virus is apparently restricted to Brazil, and occurs naturally in bidens [1], sunflower [2], yellow daisy [3] and pea [4]. Its experimental hosts include lentil, sesame, tobacco species, zinnia plants and a number of local lesion hosts [4,5].…”

“…Its experimental hosts include lentil, sesame, tobacco species, zinnia plants and a number of local lesion hosts [4,5]. BiMV shares several biological properties with Bidens mottle virus (BiMoV), a virus described in Florida infecting Asteraceae plants [6].…”