Analytica chimica acta · 2026
Unified Primer Enabled Detection (UPED) system for Magnaporthe oryzae infecting rice: A comparative study from conventional PCR to CRISPR Cas12a based detection systems.
СтатьяСравнительное исследованиеПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Karan R, Prasannakumar M K, Kour Khera Harvinder, Harish J, Patil Swathi S, Devanna Pramesh, Manjunatha C, Mishra Rakesh Kumar
Аннотация
BACKGROUND: Rice blast, caused by Magnaporthe oryzae, is one of the most devastating fungal pathogens of rice. Early, rapid and accurate detection is critical for effective disease management and the prevention of outbreaks. Conventional and isothermal molecular methods vary widely in sensitivity and field applicability, making it difficult to understand the reliable detection. This study aimed to perform the first unified, side-by-side comparison of six molecular detection techniques using a common primer set to identify the most sensitive, specific and field deployable approach for M. oryzae detection.
RESULTS: In this study, we have evaluated six diagnostic methods such as PCR, qPCR, LAMP, RPA, and CRISPR-Cas12a integrated with LAMP or RPA targeting the multi copy Pot2 transposon in M. oryzae. Sensitivity assays using 10-fold genomic DNA dilutions (109 to 10-1 copies/reaction) revealed that LAMP-CRISPR and RPA-CRISPR were most sensitive, detecting down to 1 copy/reaction. LAMP and qPCR detected down to 102 and 103 copies/reaction, while RPA and PCR were limited to 105 and 106 copies/reaction, respectively. All assays showed high specificity with no cross-reactivity to 10 non-target rice fungal pathogens. Field validation on 17 symptomatic samples confirmed that CRISPR-based methods outperformed traditional techniques, detecting positives missed by other platforms. This is the first systematic comparison applying the same primer set across multiple diagnostic methods for M. oryzae.
SIGNIFICANCE: Our findings demonstrate that CRISPR-Cas12a-based platforms combined with isothermal amplification offers high sensitivity and specificity. CRISPR based detection allows strong field applicability for M. oryzae detection. The use of a unified primer set allowed for a direct performance comparison. These results highlight the potential of CRISPR-based diagnostics to enhance early pathogen detection, enabling rapid interventions and improved management of rice blast disease.
RESULTS: In this study, we have evaluated six diagnostic methods such as PCR, qPCR, LAMP, RPA, and CRISPR-Cas12a integrated with LAMP or RPA targeting the multi copy Pot2 transposon in M. oryzae. Sensitivity assays using 10-fold genomic DNA dilutions (109 to 10-1 copies/reaction) revealed that LAMP-CRISPR and RPA-CRISPR were most sensitive, detecting down to 1 copy/reaction. LAMP and qPCR detected down to 102 and 103 copies/reaction, while RPA and PCR were limited to 105 and 106 copies/reaction, respectively. All assays showed high specificity with no cross-reactivity to 10 non-target rice fungal pathogens. Field validation on 17 symptomatic samples confirmed that CRISPR-based methods outperformed traditional techniques, detecting positives missed by other platforms. This is the first systematic comparison applying the same primer set across multiple diagnostic methods for M. oryzae.
SIGNIFICANCE: Our findings demonstrate that CRISPR-Cas12a-based platforms combined with isothermal amplification offers high sensitivity and specificity. CRISPR based detection allows strong field applicability for M. oryzae detection. The use of a unified primer set allowed for a direct performance comparison. These results highlight the potential of CRISPR-based diagnostics to enhance early pathogen detection, enabling rapid interventions and improved management of rice blast disease.