RESUMEN
The pathogen Colletotrichum siamense causes tea anthracnose, resulting in economic losses to the Chinese tea industry. To effectively diagnose this pathogen in the field, we developed a loop-mediated isothermal amplification (LAMP) method using highly specific primers with a sensitivity of 1 pg/µl designed for amplifying the CAL gene, which was 10 times higher than that of conventional PCR. Additionally, to improve the method for obtaining DNA samples required for on-site diagnosis, we used the filter-disc DNA extraction method, which does not require special instruments and can be completed in a few minutes, and found that it effectively meets the requirements for the LAMP reaction. Finally, we combined LAMP with a filter-disc DNA extraction method (FDE-LAMP) to diagnose different degrees of disease in inoculated samples and 20 samples from the field. The results showed that the procedure had sufficient sensitivity for pathogen detection. Therefore, the FDE-LAMP procedure could greatly contribute to managing and preventing tea anthracnose in the field.
Asunto(s)
Colletotrichum , ADN , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Té , Sensibilidad y EspecificidadRESUMEN
The poor fidelity of T4 DNA ligase has always limited the simple detection of single-nucleotide polymorphisms (SNPs) and is only applicable to some special SNP types. This study developed a highly sensitive and specific detection method for SNPs based on high-fidelity single-stranded circularisation. It used T4 DNA ligase and rolling circle amplification (RCA) plus loop-mediated isothermal amplification (LAMP). Surprisingly, the cyclisation stage's efficiency greatly improved. The ligation fidelity was almost perfect via the unique pairing pattern between a long-paired base at the 5' terminus and only five bases at the 3' terminus on linear single-stranded DNA (l-DNA). Subsequently, LR-LAMP was performed and combined with the circularisation step for the simple detection of SNPs. The results showed that even 100 aM targets could be detected correctly and that a mutation rate of 0.1% or even 0.01% could be analysed via naked-eye visualisation or fluorescence detection, respectively. In addition, genomic DNA samples were used to evaluate the method, which indicated that it could effectively distinguish the SNPs of RPA190-T1145A in Phytophthora infestans. This strategy may play an important role in both circularisation of single-stranded DNA and detecting arbitrary SNPs.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico , Polimorfismo de Nucleótido Simple , ADN/genética , ADN Ligasas , ADN de Cadena Simple/genética , Genotipo , Técnicas de Amplificación de Ácido Nucleico/métodosRESUMEN
Correction for 'High-efficiency and high-fidelity ssDNA circularisation via the pairing of five 3'-terminal bases to assist LR-LAMP for the genotyping of single-nucleotide polymorphisms' by Taiwen Li et al., Analyst, 2022, https://doi.org/10.1039/d2an01042a.
RESUMEN
BACKGROUND: The on-site molecular detection of plant pathogens is particularly important for the development of sustainable agriculture. Extracting DNA from plant tissues, microbes or coexisting environments is complex, labor-intensive and time-consuming. To facilitate this process, we propose a DNA purification strategy based on graphene oxide (GO). RESULTS: The excellent adsorption ability of GO was verified by visualizing changes in its microscopic surface and macroscopic mixture. To further optimize the DNA purification, we determined the optimal GO concentration and treatment time at 95 °C (2 mg mL-1 and 2 min, respectively). We confirmed that our strategy is effective on plant tissues and various microorganisms, and that the obtained DNA can be directly used for polymerase chain reaction amplification. Combining the proposed GO-based DNA purification method with the loop-mediated isothermal amplification method is superior, in terms of the required steps, time, cost and detection effect, to the cetyltrimethylammonium bromide method and a commercial kit for detecting plant pathogens. CONCLUSION: We present a feasible, rapid, simple and low-cost DNA purification method with high practical value for scientific applications in plant pathogen detection. This strategy can also provide important technical support for future research on plant-microbial microenvironments. © 2024 Society of Chemical Industry.
Asunto(s)
Grafito , Grafito/química , Técnicas de Amplificación de Ácido Nucleico/métodos , Enfermedades de las Plantas/microbiología , ADN Bacteriano , Reacción en Cadena de la Polimerasa/métodos , Adsorción , Técnicas de Diagnóstico MolecularRESUMEN
Globisporangium, especially G. sylvaticum, causes devastating root rot, blight, and other diseases in various species of cash crops. To investigate the distribution and host range of G. sylvaticum in Guizhou, a suitable habitat for this pathogen, we collected 156 root-diseased samples, isolated the pathogens, and found that G. sylvaticum is widespread and has eleven host plants, including four novel hosts. Furthermore, to effectively identify G. sylvaticum, we developed a simple and dependable method based on loop-mediated isothermal amplification (LAMP), which used a primer set designed from the internal transcribed spacer sequences with high specificity and sensitivity of 1 pg/µL. Additionally, to perform field identification, we used the "Plant-LAMP" method with crude DNA extraction to detect the pathogen in 45 root samples from nine species of plants. Our results showed that this method could effectively detect G. sylvaticum in diseased roots. Therefore, our findings not only enrich existing research on the diversity of pathogenic Globisporangium in Guizhou but also present an efficient LAMP field detection method that could significantly contribute to plant disease management and prevention.