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

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

10.21203/rs.3.rs-96229/v2 ◽

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

10.21203/rs.3.rs-96229/v1 ◽

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.

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A Novel Lateral Flow Assay for Rapid and Sensitive Nucleic Acid Detection of Avibacterium paragallinarum

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.738558 ◽

2021 ◽

Vol 8 ◽

Author(s):

Caiyun Huo ◽

Donghai Li ◽

Zhenguo Hu ◽

Guiping Li ◽

Yanxin Hu ◽

...

Keyword(s):

Nucleic Acid ◽

Gel Electrophoresis ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Nucleic Acid Hybridization ◽

Nucleic Acid Detection ◽

Agarose Gel ◽

Agarose Gel Electrophoresis ◽

Infectious Coryza ◽

Avibacterium Paragallinarum

Avibacterium paragallinarum, the pathogen of infectious coryza, caused a highly contagious respiratory disease that poses a serious threat to chickens. Hence, it is necessary to do diagnostic screening for Av. paragallinarum. Existing technologies have been used for Av. paragallinarum testing, which, however, have some drawbacks such as time consuming and expensive that require well-trained personnel and sophisticated infrastructure, especially when they are limitedly feasible in some places for lack of resources. Nucleic acid hybridization-based lateral flow assay (LFA) is capable of dealing with these drawbacks, which is attributed to the advantages, such low cost, rapid, and simple. However, nucleic acid determination of Av. paragallinarum through LFA method has not been reported so far. In this study, we developed a novel LFA method that employed gold nanoparticle probes to detect amplified Av. paragallinarum dsDNA. Compared with agarose gel electrophoresis, this LFA strip was inexpensive, simple- to- use, and time- saving, which displayed the visual results within 5–8 min. This LFA strip had higher sensitivity that achieved the detection limit of 101 CFU/ml compared with 102 CFU/ml in agarose gel electrophoresis. Besides, great sensitivity was also shown in the LFA strip, and no cross reaction existed for other bacteria. Furthermore, Av. paragallinarum in clinical chickens with infectious coryza were perfectly detected by our established LFA strip. Our study is the first to develop the LFA integrated with amplification and sample preparation techniques for better nucleic acid detection of Av. paragallinarum, which holds great potential for rapid, accurate, and on-site determination methods for early diagnosis of Av. paragallinarum to control further spreading.

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Improved Analytical Sensitivity of Lateral Flow Assay using Sponge for HBV Nucleic Acid Detection

Scientific Reports ◽

10.1038/s41598-017-01558-x ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 34

Author(s):

Ruihua Tang ◽

Hui Yang ◽

Yan Gong ◽

Zhi Liu ◽

XiuJun Li ◽

...

Keyword(s):

Nucleic Acid ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Nucleic Acid Detection ◽

Analytical Sensitivity

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Direct Nucleic Acid Detection of Diarrheal Pathogens in Stool Using an Antibody-Free Lateral Flow Assay

Gastroenterology ◽

10.1016/s0016-5085(17)32826-3 ◽

2017 ◽

Vol 152 (5) ◽

pp. S817

Author(s):

Nicolaas H. Fourie ◽

Sarah K. Abey ◽

Eric Ferguson ◽

Natnael Kenea ◽

Ana F. Diallo ◽

...

Keyword(s):

Nucleic Acid ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Nucleic Acid Detection

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Fluorescent Probe-Based Lateral Flow Assay for Multiplex Nucleic Acid Detection

Analytical Chemistry ◽

10.1021/ac5010458 ◽

2014 ◽

Vol 86 (12) ◽

pp. 5611-5614 ◽

Cited By ~ 84

Author(s):

Ye Xu ◽

Yinghua Liu ◽

Yan Wu ◽

Xiaohu Xia ◽

Yiqun Liao ◽

...

Keyword(s):

Nucleic Acid ◽

Fluorescent Probe ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Nucleic Acid Detection

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Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.613304 ◽

2021 ◽

Vol 11 ◽

Author(s):

Yu-Zhong Zheng ◽

Jiang-Tao Chen ◽

Jian Li ◽

Xian-Jing Wu ◽

Jin-Zhou Wen ◽

...

Keyword(s):

Reverse Transcription ◽

Influenza A ◽

Limit Of Detection ◽

Lateral Flow ◽

Rapid Screening ◽

N Gene ◽

Clinical Samples ◽

Influenza B ◽

Lateral Flow Dipstick ◽

Human Coronaviruses

BackgroundThe emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally. With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed.MethodsTargeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed. For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively. For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA. For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested via quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively.ResultsThe limit of detection was 1 copies/μl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens. Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples.ConclusionThis work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas.

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Rapid Point-of-Care Testing for SARS-CoV-2 Virus Nucleic Acid Detection by an Isothermal and Nonenzymatic Signal Amplification System Coupled with a Lateral Flow Immunoassay Strip

Sensors and Actuators B Chemical ◽

10.1016/j.snb.2021.129899 ◽

2021 ◽

pp. 129899

Author(s):

Mingyuan Zou ◽

Feiya Su ◽

Rui Zhang ◽

Xinglu Jiang ◽

Han Xiao ◽

...

Keyword(s):

Nucleic Acid ◽

Signal Amplification ◽

Point Of Care ◽

Lateral Flow ◽

Lateral Flow Immunoassay ◽

Nucleic Acid Detection ◽

Point Of Care Testing ◽

Amplification System ◽

Signal Amplification System

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Circulation of Respiratory Viruses in Hospitalized Adults before and during the COVID-19 Pandemic in Brescia, Italy: A Retrospective Study

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18189525 ◽

2021 ◽

Vol 18 (18) ◽

pp. 9525

Author(s):

Maria Antonia De Francesco ◽

Caterina Pollara ◽

Franco Gargiulo ◽

Mauro Giacomelli ◽

Arnaldo Caruso

Keyword(s):

Respiratory Syncytial Virus ◽

Influenza A ◽

Statistical Significance ◽

Respiratory Viruses ◽

Influenza Viruses ◽

Contact Tracing ◽

Nucleic Acid Detection ◽

Respiratory Pathogens ◽

Syncytial Virus ◽

Human Coronaviruses

Different preventive public health measures were adopted globally to limit the spread of SARS-CoV-2, such as hand hygiene and the use of masks, travel restrictions, social distance actions such as the closure of schools and workplaces, case and contact tracing, quarantine and lockdown. These measures, in particular physical distancing and the use of masks, might have contributed to containing the spread of other respiratory viruses that occurs principally by contact and droplet routes. The aim of this study was to evaluate the prevalence of different respiratory viruses (influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses 1, 2, 3 and 4, rhinovirus, adenovirus, metapneumovirus and human coronaviruses) after one year of the pandemic. Furthermore, another aim was to evaluate the possible impact of these non-pharmaceutical measures on the circulation of seasonal respiratory viruses. This single center study was conducted between January 2017–February 2020 (pre-pandemic period) and March 2020–May 2021 (pandemic period). All adults >18 years with respiratory symptoms and tested for respiratory pathogens were included in the study. Nucleic acid detection of all respiratory viruses was performed by multiplex real time PCR. Our results show that the test positivity for influenza A and B, metapneumovirus, parainfluenza virus, respiratory syncytial virus and human coronaviruses decreased with statistical significance during the pandemic. Contrary to this, for adenovirus the decrease was not statistically significant. Conversely, a statistically significant increase was detected for rhinovirus. Coinfections between different respiratory viruses were observed during the pre-pandemic period, while the only coinfection detected during pandemic was between SARS-CoV-2 and rhinovirus. To understand how the preventive strategies against SARS-CoV-2 might alter the transmission dynamics and epidemic patterns of respiratory viruses is fundamental to guide future preventive recommendations.

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