RESUMO
A rapid method for detection, discrimination and quantification of wheat and barley strains of wheat dwarf virus (WDV) was successfully developed. The sensitivity of quantification of the wheat and barley strains of WDV ranged from an average of 1.2 × 10(7)-1.2 × 10(2) and from an average of 1.4 × 10(7)-1.4 × 10(4) copies of viral genome, respectively. These standard serial dilutions were applied to plant and vector tissues for virus titer calculations. Both strains of WDV were clearly discriminated by specific probes and melting curve analysis. Both TaqMan(®) and SYBR(®) Green technologies provided accurate and reliable methods for monitoring, detection, discrimination, and quantification of WDV.
Assuntos
Geminiviridae/classificação , Geminiviridae/isolamento & purificação , Hordeum/virologia , Doenças das Plantas/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Triticum/virologia , Carga Viral/métodos , Geminiviridae/genética , Sensibilidade e EspecificidadeRESUMO
A SYBR Green(®)-based one step RT-qPCR assay was developed for the detection and quantification of Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV). The RT-qPCR assay employed seven plant-expressed genes-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA, ubiquitin, ribosomal protein S19, Rubisco, RNA polymerase subunit II and ß-actin-as internal reference housekeeping genes in a relative quantification system in three apple cultivars (i.e. Idared, Champion, Fragrance). The average expression stability (M) found by GeNorm software suggest that GAPDH and S19 were the most stable reference genes. We propose employing GAPDH and S19 as housekeeping genes for accurate quantification of ASGV and ApMV in apple leaf samples. The detection limit for both viruses was found around 70 copies of viral genome by one-step RT-qPCR.
RESUMO
Single-strand conformation polymorphism (SSCP) and low-stringency single specific primer (LSSP)-PCR were assessed for suitability and reliability in genotyping of Plum pox virus (PPV) isolates. Examined PPV isolates included 16 PPV-D, 12 PPV-M, and 14 PPV-Rec isolates collected in Czech Republic. The analysis was performed on the helper component protease (HC-Pro) region of the PPV genome. SSCP and LSSP-PCR allowed the differentiation of PPV strain, but SSCP was not able to distinguish isolates within the same strain. The individual genotyping of each PPV isolate was obtained by LSSP-PCR. Nevertheless, both SSCP and LSSP-PCR techniques are suitable for preliminary screening of genetic variability of plant RNA viruses.
Assuntos
Cisteína Endopeptidases/genética , Vírus Eruptivo da Ameixa/classificação , Vírus Eruptivo da Ameixa/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético , RNA Viral/genética , Proteínas Virais/genética , República Tcheca , Vírus Eruptivo da Ameixa/isolamento & purificação , Polimorfismo Conformacional de Fita SimplesRESUMO
Cereal-infecting Mastreviruses are one of the most ubiquitous of viruses, having caused huge yield losses during the last decade in the Czech Republic. The presence of two strains of Wheat dwarf virus (WDV), one of which is wheat adapted and one barley adapted, have been confirmed from among field samples of wheat and barley plants. The virus typing was conducted by both PCR-RFLP and sequencing-based methods. The Czech WDV isolates of the barley strain are more variable than the isolates of the wheat strain, separating them into two clades, one representing a pool of divergent isolates. The RFLP analysis confirmed the separation of the Czech isolates into two strains and highlights a powerful method for the rapid diagnosis of both the wheat and barley strains. Additionally, both RFLP and sequence analysis have shown that the barley strain is restricted to the barley host, while the wheat strain is present in both wheat and barley plants.
