Dataset Information

Optimization and Validation of Reverse Transcription Recombinase-Aided Amplification (RT-RAA) for Sorghum Mosaic Virus Detection in Sugarcane.

ABSTRACT: Sorghum mosaic virus (SrMV) causes sugarcane mosaic disease and has significant adverse economic impacts on the cultivation of sugarcane. This study aimed to develop a rapid isotherm nucleic acid amplification method for detecting SrMV. Specific primers were designed to target the conserved region of the P3 gene of SrMV. The reverse transcription recombinase-aided amplification (RT-RAA) method was developed by screening primers and optimizing reaction conditions. Comparative analyses with RT-PCR demonstrated that the RT-RAA method exhibited superior specificity, sensitivity, and reliability for SrMV detection. Notably, using a standard plasmid diluted 10-fold continuously as a template, the sensitivity of RT-RAA was 100-fold higher than that of RT-PCR. Moreover, the RT-RAA reaction displayed flexibility in a temperature range of 24-49 °C, eliminating the need for expensive and complex temperature control equipment. Thus, this method could be utilized at ambient or even human body temperature. Within a short duration of 10 min at 39 °C, the target sequence of SrMV could be effectively amplified. Specificity analysis revealed no cross-reactivity between SrMV and other common sugarcane viruses detected via the RT-RAA. With its high sensitivity, rapid reaction time, and minimal equipment requirements, this method presents a promising diagnostic tool for the reliable and expedited detection of SrMV. Furthermore, it indicates broad applicability for successfully detecting other sugarcane viruses.

SUBMITTER: Wang F

PROVIDER: S-EPMC10457762 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Publications

Optimization and Validation of Reverse Transcription Recombinase-Aided Amplification (RT-RAA) for Sorghum Mosaic Virus Detection in Sugarcane.

Wang Fenglin F Liang Qinmin Q Lv Rongman R Ahmad Shakeel S Ahmad Shakeel S Bano Mishal M Weng Guangzhen G Wen Ronghui R

Pathogens (Basel, Switzerland) 20230818 8

Sorghum mosaic virus (SrMV) causes sugarcane mosaic disease and has significant adverse economic impacts on the cultivation of sugarcane. This study aimed to develop a rapid isotherm nucleic acid amplification method for detecting SrMV. Specific primers were designed to target the conserved region of the P3 gene of SrMV. The reverse transcription recombinase-aided amplification (RT-RAA) method was developed by screening primers and optimizing reaction conditions. Comparative analyses with RT-PCR ...[more]

PMID: 37624015

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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

Optimization and Validation of Reverse Transcription Recombinase-Aided Amplification (RT-RAA) for Sorghum Mosaic Virus Detection in Sugarcane.

Publications

Optimization and Validation of Reverse Transcription Recombinase-Aided Amplification (RT-RAA) for Sorghum Mosaic Virus Detection in Sugarcane.

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