Development of a method for the specific diagnosis of PepMoV based on RT -PCR

Revista de Protecci贸n Vegetal

Pacheco Sánchez, Ronald¹  Piñol Pérez, Bertha²  Díaz De la Osa, Acela¹  Arana Labrada, Franklyn³  Quiñones Pantoja, Madelaine² 

[1] Universidad de La Habana, Cuba;Centro Nacional de Sanidad Agropecuaria (CENSA), San José de las Lajas, Cuba;Universidad de Las Tunas, Las Tunas, Cuba

关键词: RT-PCR;    potyviruses;    PepMoV;    sweet pepper;   

DOI  :  

学科分类：农业科学（综合）

来源: Centro Nacional de Sanidad Agropecuaria

【 摘 要 】

The main objective was to develop a method for the specific diagnosis of PepMoV based on RT-PCR. Primers were designed using as reference the genomic sequence of the Florida isolate to amplify a fragment of the 5'untranslatable end and amino-terminus of P1 protein of the genome. The Oligo program (version 7.4) was used to carry out the analysis in silico and establish the initial parameters of the reaction. The optimal temperature of primer hybridization, primer concentration, intra- and inter-assay repeatability, detection limit, and the analytic specificity were evaluated. The optimal temperature of primer hybridization was 59°C, and 0,32 µM was selected as the optimal concentration of the primers. The aspects of the assay evaluated allowed establishing the optimal conditions of the technique. The RT-PCR method behaved with a high specificity for the diagnosis of PepMoV. It showed a detection limit of 94 pg.µl-1. The molecular tool obtained will allow supporting the breeding programs and the integrated pest management of this crop in the country.

【 授权许可】

CC BY-NC