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

2021 ◽  
Vol 9 ◽  
Author(s):  
Xiao Fu Wang ◽  
Wen Qiang Chen ◽  
Jian Li Guo ◽  
Cheng Peng ◽  
Xiao Yun Chen ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Dna Extraction ◽  
Molecular Detection ◽  
Future Research ◽  
Nucleic Acid Detection ◽  
Valuable Insight ◽  
Detection Techniques ◽  
Detection Process ◽  
Novel Approach ◽  
Chemical Heating

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.

scholarly journals One-Tube SARS-CoV-2 Detection Platform Based on RT-RPA and CRISPR/Cas12a

2020 ◽  
Author(s):  
Yangyang Sun ◽  
Lei Yu ◽  
Chengxi Liu ◽  
Wei Chen ◽  
Dechang Li ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
N Gene ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Detection Process ◽  
Human Coronaviruses ◽  
Negative Controls

Abstract Background: COVID-19 has spread rapidly around the world, affecting almost every person. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component. Methods: We designed RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) primers of RdRp gene and N gene according to the SARS-CoV-2 gene sequence. We optimized the components in the reaction so that the detection process could be carried out in one tube. The specificity was demonstrated through detecting nucleic acid samples from seven human coronaviruses. Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards diluted by different gradients were used to demonstrate the limit of detection. Furthermore, we have developed a lateral flow assay based on OR-DETECTR for the detection of COVID-19. Results: We have developed a o ne-tube detection platform based on R T- R PA and DNA Endonuclease-Targeted CRISPR Trans Reporter ( DETECTR ) technology, termed OR-DETECTR, to detect SARS-CoV-2. The detection process is completed in one tube, and the time is 50min. The method can specifically detect SARS-CoV-2 from seven human coronaviruses with a low detection limit of 2.5 copies/µl input. Results from six SARS-CoV-2 patient samples, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. Furthermore, we have developed a lateral flow assay based on OR-DETECTR for the detection of COVID-19. Conclusions: OR-DETECTR detection platform is rapid, accurate, tube closed, easy-to-operate, and free of large instruments for COVID-19 detection.

scholarly journals CRISPR-Assisted DNA Detection, a novel dCas9-based DNA detection technique

2020 ◽  
Author(s):  
Xinhui Xu ◽  
Tao Luo ◽  
Jinliang Gao ◽  
Na Lin ◽  
Weiwei Li ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Room Temperature ◽  
Dna Detection ◽  
Point Of Care ◽  
Hpv Infection ◽  
Solid Support ◽  
Human Papillomaviruses ◽  
Nucleic Acid Detection ◽  
Detection Techniques ◽  
Signal Readout

AbstractNucleic acid detection techniques are always critical to diagnosis, especially in the background of the present COVID-19 pandemic. The simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, the current nucleic acid detection techniques are still limited the traditional amplification and hybridization. To overcome the limitation, we here develop a CRISPR/Cas9-assisted DNA detection (CADD). In this detection, DNA sample is incubated with a pair of capture sgRNAs (sgRNAa and sgRNAb) specific to a target DNA, dCas9, a signal readout-related probe, and an oligo-coated solid support beads or microplate at room temperature for 15 min. During this incubation, the dCas9-sgRNA-DNA complex is formed and captured on solid support by the capture sequence of sgRNAa and the signal readout-related probe is captured by the capture sequence of sgRNAb. Finally the detection result is reported by a fluorescent or colorimetric signal readout. This detection was verified by detecting DNA of bacteria, cancer cell and virus. Especially, by designing a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), the HPV infection in 64 clinical cervical samples were successfully detected by the method. All detections can be finished in 30 minutes at room temperature. This detection holds promise for rapid on-the-spot detection or point-of-care testing (POCT).

scholarly journals One-tube SARS-CoV-2 detection platform based on RT-RPA and CRISPR/Cas12a

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yangyang Sun ◽  
Lei Yu ◽  
Chengxi Liu ◽  
Shanting Ye ◽  
Wei Chen ◽  
...  
Keyword(s):  
Detection Limit ◽  
Nucleic Acid ◽  
Influenza A ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Detection Process ◽  
Influenza A H1n1 ◽  
Human Coronaviruses

Abstract Background COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component. Methods We tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19. Results The OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input. Conclusions The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.

scholarly journals One-tube SARS-CoV-2 detection platform based on RT-RPA and CRISPR/Cas12a

2021 ◽  
Author(s):  
Yangyang Sun ◽  
Lei Yu ◽  
Chengxi Liu ◽  
Shanting Ye ◽  
Wei Chen ◽  
...  
Keyword(s):  
Detection Limit ◽  
Nucleic Acid ◽  
Influenza A ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Detection Process ◽  
Influenza A H1n1 ◽  
Human Coronaviruses

Abstract Background: COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component. Methods: We tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19.Results: The OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input.Conclusions: The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.

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