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A Fast, Visual, and Instrument-free Platform Involving Rapid DNA Extraction, Chemical Heating, and Recombinase Aided Amplification for On-Site Nucleic Acid Detection.

ABSTRACT: The nucleic acid-based technique has been widely utilized in many fields including for on-site detection. However, traditional molecular detection techniques encounter limitations like relying on instruments, time consuming or complex operation, and cannot meet the demands of on-site testing. In this study, a rapid DNA extraction method (RDEM), recombinase aided amplification (RAA), and chemical heating packet (CHP) are integrated and termed as RRC platform for on-site detection of nucleic acid. For demonstration purposes, SHZD32-1 (a new transgenic soybean line from China) was detected using the novel platform to demonstrate its feasibility and capability for on-site detection. Using the RDEM, high-quality DNA appropriate for molecular detection was quickly extracted in 3-5 min. The heat energy generated by CHP was met the temperature requirements of RAA. Using the RRC platform, the whole detection process can be accomplished within only 30 min, and the results can be visually detected with glasses under blue light. No special or expensive instrument was needed for the detection process. This study provides a novel approach for on-site detection of nucleic acids besides providing valuable insight on related future research.

SUBMITTER: Wang XF

PROVIDER: S-EPMC8645684 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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/?L, 10 times higher than that of the conventional PCR method (103 copies/?L); the RF-RT-RAA method could be used to complete reaction within 26 min, and its lowest detectable limit was 10 copies/?L, 100 times higher than that of conventional PCR method (103 copies/?L), and it was as sensitive as real-time fluorescence-based quantitative PCR (10 copies/?L). 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.

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A Fast, Visual, and Instrument-free Platform Involving Rapid DNA Extraction, Chemical Heating, and Recombinase Aided Amplification for On-Site Nucleic Acid Detection.

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