Dataset Information

On-site and visual detection of sorghum mosaic virus and rice stripe mosaic virus based on reverse transcription-recombinase-aided amplification and CRISPR/Cas12a.

ABSTRACT: Rapid, sensitive and visual detection of plant viruses is conducive to effective prevention and control of plant viral diseases. Therefore, combined with reverse transcription and recombinase-aided amplification, we developed a CRISPR/Cas12a-based visual nucleic acid detection system targeting sorghum mosaic virus and rice stripe mosaic virus, which cause harm to crop production in field. When the RT-RAA products were recognized by crRNA and formed a complex with LbCas12a, the ssDNA labeled with a quenched green fluorescent molecule will be cleaved by LbCas12a, and then a significant green fluorescence signal will appear. The entire detection process can be completed within 30 min without using any sophisticated equipment and instruments. The detection system could detect samples at a dilution of 10⁷, about 10⁴-fold improvement over RT-PCR, so the system was successfully to detect rice stripe mosaic virus in a single leafhopper, which is the transmission vector of the virus. Finally, the CRISPR/Cas12a-based detection system was utilized to on-site detect the two viruses in the field, and the results were fully consistent with that we obtained by RT-PCR in laboratory, demonstrating that it has the application prospect of detecting important crop viruses in the field.

SUBMITTER: Wang J

PROVIDER: S-EPMC9895793 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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On-site and visual detection of sorghum mosaic virus and rice stripe mosaic virus based on reverse transcription-recombinase-aided amplification and CRISPR/Cas12a.

Wang Junkai J Huang Xiuqin X Chen Siping S Chen Jiahao J Liang Zhengyi Z Chen Biao B Yang Xin X Zhou Guohui G Zhang Tong T

Frontiers in genome editing 20230120

Rapid, sensitive and visual detection of plant viruses is conducive to effective prevention and control of plant viral diseases. Therefore, combined with reverse transcription and recombinase-aided amplification, we developed a CRISPR/Cas12a-based visual nucleic acid detection system targeting sorghum mosaic virus and rice stripe mosaic virus, which cause harm to crop production in field. When the RT-RAA products were recognized by crRNA and formed a complex with LbCas12a, the ssDNA labeled with ...[more]

PMID: 36741944

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Project description:Newcastle disease is an acute and highly contagious disease of poultry caused by Newcastle disease virus infection, which does great harm to the poultry industry all over the world. To diagnose the disease simply and quickly, 2 detection methods were established based on reverse transcription recombinase-aided amplification (RT-RAA) technology. One is reverse transcription recombinase-aided amplification-lateral flow dipstick (RT-RAA-LFD) that is to combine RT-RAA with lateral flow dipstick; the other is real-time fluorescence-based reverse transcription recombinase-aided amplification (RF-RT-RAA) that is the combination of RT-RAA and exo probe. In this study, the reaction conditions such as reaction temperature and reaction time of the 2 methods were optimized, and their specificity and sensitivity were tested. The results showed that the RT-RAA-LFD method could be used to complete reaction within 23 min, and its lowest detectable limit was 102 copies/μL, 10 times higher than that of the conventional PCR method (103 copies/μL); the RF-RT-RAA method could be used to complete reaction within 26 min, and its lowest detectable limit was 10 copies/μL, 100 times higher than that of conventional PCR method (103 copies/μL), and it was as sensitive as real-time fluorescence-based quantitative PCR (10 copies/μL). The 2 methods had no cross reaction to the nucleic acid of other avian pathogens and showed good specificity. A total of 86 clinical samples suspected of the Newcastle disease virus were tested by conventional PCR, real-time fluorescence-based quantitative PCR, RT-RAA-LFD, and RF-RT-RAA. Based on the commonly used conventional PCR method, the other 3 detection methods had a coincidence rate of higher than 93%. In summary, RT-RAA-LFD and RF-RT-RAA had high specificity, sensitivity, and efficiency, which were suitable for clinical and laboratory diagnosis, respectively, and provided technical support for the prevention and control of Newcastle disease.

Dataset Information

On-site and visual detection of sorghum mosaic virus and rice stripe mosaic virus based on reverse transcription-recombinase-aided amplification and CRISPR/Cas12a.

Publications

On-site and visual detection of sorghum mosaic virus and rice stripe mosaic virus based on reverse transcription-recombinase-aided amplification and CRISPR/Cas12a.

Similar Datasets

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