RESUMO
A differential detection reverse transcription loop-mediated isothermal amplification (DD-RT-LAMP) method was developed to detect either Barley yellow mosaic virus (BaYMV) or Japanese soil-borne wheat mosaic virus (JSBWMV) simultaneously. Both primer sets, which recognized either BaYMV or JSBWMV genomic RNA, amplified DNA more efficiently at 65°C using an isothermal DNA amplification and fluorescence detection device. Furthermore, these primer sets showed unique annealing curves. The peak annealing temperatures of BaYMV and JSBWMV amplification products using specific primer sets were 86.9°C-87.7°C and 84.5°C-85.0°C, respectively, and were clearly distinguishable during an annealing step following the isothermal amplification, monitored using a fluorescence detection device. In the field samples of barley (Hordeum vulgare L.) tested, BaYMV or JSBWMV were detected by DD-RT-LAMP, and the detection results of DD-RT-LAMP were correspondent with the results of reverse transcription-PCR.
Assuntos
Hordeum , Vírus de Plantas , Transcrição Reversa , Hordeum/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Sensibilidade e Especificidade , Doenças das Plantas/virologia , Vírus de Plantas/isolamento & purificaçãoRESUMO
For a simple and rapid detection of Chrysanthemum stem necrosis virus (CSNV) from chrysanthemum and tomato, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed. A primer set designed to the genome sequences of CSNV worked most efficiently at 63°C and could detect CSNV RNA within 12min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. The result of a specificity test using seven other viruses and one viroid-infectable chrysanthemum or tomato showed that the assay could amplify CSNV specifically, and a sensitivity comparison showed that the RT-LAMP assay was as sensitive as the reverse transcriptase polymerase chain reaction. The RT-LAMP assay using crude RNA, extracted simply, could detect CSNV. Overall, the RT-LAMP assay was found to be a simple, specific, convenient, and time-saving method for CSNV detection.
Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças das Plantas/virologia , Vírus de Plantas/isolamento & purificação , Vírus de RNA/isolamento & purificação , Chrysanthemum , Primers do DNA/genética , Fluorescência , Solanum lycopersicum , RNA Viral/genética , Sensibilidade e Especificidade , TemperaturaRESUMO
Root rot of poinsettia, caused by Pythium helicoides at high temperatures in hydroponic cultures, has become a serious problem in many parts of the world. We have developed a species-specific, loop-mediated isothermal amplification (LAMP) assay for the rapid diagnosis of this pathogen. The primers were designed using the ribosomal DNA internal transcribed spacer sequence. Primer specificity was established using 40 Pythium species including P. helicoides, 11 Phytophthora species, and eight other soil-borne pathogens. A sensitivity test was carried out using genomic DNA extracted from P. helicoides, and the detection limit was c. 100 fg which is comparable to that of the polymerase chain reaction (PCR). In addition, we tested the ease of pathogen detection in poinsettia roots. The LAMP results were consistent with those from the conventional plating method and showed more sensitivity than the PCR results. Consequently, the LAMP method developed in this study is effective for the rapid and easy detection of P. helicoides.
Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Oomicetos/isolamento & purificação , Primers do DNA/genética , DNA Espaçador Ribossômico/genética , Euphorbia/microbiologia , Oomicetos/genética , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , Sensibilidade e EspecificidadeRESUMO
A differential detection method for three wheat viruses: Wheat yellow mosaic virus (WYMV), Japanese soil-borne mosaic virus (JSBWMV) and Chinese wheat mosaic virus (CWMV) using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction was developed. All three primer sets, which were designed from the genome sequences of WYMV, JSBWMV and CWMV respectively, worked most efficiently at 65 °C and could detect each virus RNA within 10 min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. Furthermore, these primer sets showed unique annealing curves. The peak denaturing temperatures of WYMV, JSBWMV and CWMV primer sets were 87.6 °C, 84.8 °C and 86.4 °C, respectively and were clearly distinguished by the isothermal DNA amplification and fluorescence detection device. The RT-LAMP assay including all three primer sets was found to be 100 times more sensitive than RT-PCR for WYMV and JSBWMV and as sensitive as RT-PCR for CWMV. The RT-LAMP method was validated for the simultaneous detection of these viruses in wheat and barley leaves.
Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças das Plantas/virologia , Vírus de Plantas/isolamento & purificação , Vírus de RNA/isolamento & purificação , Transcrição Reversa , Triticum/virologia , Primers do DNA/genética , Vírus de Plantas/genética , Vírus de RNA/genética , Sensibilidade e EspecificidadeRESUMO
In this study, LAMP markers linked to shelf-life in melon (Cucumis melo L.) were developed by converting a cleaved amplified polymorphic sequences (CAPS) marker (C2). The CAPS-PCR fragments from the long-shelf-life melon (O-3) and short-shelf-life melon (Nat-2) were cloned and sequenced to construct LAMP primers. A single nucleotide polymorphism (SNP) was identified between O-3 and Nat-2. LAMP primers were designed to detect the SNP. In the LAMP reaction to detect long-shelf-life melon, the turbidity of the templates using O-3, F1, homozygous long-shelf-life F2 lines and heterozygous long-shelf-life F2 lines started to increase after 40 min. In contrast, the turbidity of Nat-2 and homozygous short-shelf-life F2 lines did not increase even after 90 min. In the LAMP reaction to detect short-shelf-life melon, the turbidity of the templates using Nat-2, F1, homozygous short-shelf-life F2 lines and heterozygous long-shelf-life F2 lines started to increase after 40 min. But the turbidity of O-3 and homozygous long-shelf-life F2 lines did not increase after 90 min. This attests to the high reliability and usefulness of LAMP for marker-assisted selection.
Assuntos
Cucumis melo/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Polimorfismo de Nucleotídeo Único , Sequência de Bases , Clonagem Molecular , Cruzamentos Genéticos , Cucumis melo/metabolismo , Tecnologia de Alimentos , Marcadores Genéticos , Heterozigoto , Homozigoto , Dados de Sequência MolecularRESUMO
An immunocapture reverse transcription loop-mediated isothermal amplification (IC/RT-LAMP) was developed for the detection of tomato spotted wilt virus (TSWV) from chrysanthemum. This method enabled sensitive, reproducible and specific detection of TSWV from chrysanthemum plants. In the RT-LAMP method, TSWV genomic RNA could be amplified under isothermal (65 degrees C) conditions within 1 h. The resulting amplicons were detected by the measurement or observation of the turbidity of the reaction mixture without gel electrophoresis. IC/RT-LAMP was 100 times more sensitive than IC/RT-PCR.
Assuntos
Chrysanthemum/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças das Plantas/virologia , Tospovirus/isolamento & purificação , Sequência de Bases , Dados de Sequência Molecular , Nefelometria e Turbidimetria , RNA Viral/análise , RNA Viral/isolamento & purificação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tospovirus/genéticaRESUMO
The genomic DNA molecule of tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted geminivirus, was amplified from total DNA extracts of TYLCV-infected tomato (Lycopersicon esculentum) by the use of loop-mediated isothermal amplification (LAMP). The procedure was also used to amplify TYLCV DNA from total DNA extracts of individual whiteflies (Bemisia tabaci) that had fed on TYLCV-infected plants. One of the characteristics of the LAMP method is its ability to synthesize an extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced yielding a white precipitate of magnesium pyrophosphate in the reaction mixture. The presence or absence of this white precipitate allows easy detection of amplification of TYLCV genomic DNA without gel electrophoresis.