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

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.

Download Full-text

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.

Download Full-text

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

Download Full-text

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

Journal of Translational Medicine ◽

10.1186/s12967-021-02741-5 ◽

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.

Download Full-text

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

Download Full-text

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.

Download Full-text

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

Download Full-text

Development of a Generic Microfluidic Device for Simultaneous Detection of Antibodies and Nucleic Acids in Oral Fluids

BioMed Research International ◽

10.1155/2013/543294 ◽

2013 ◽

Vol 2013 ◽

pp. 1-12 ◽

Cited By ~ 10

Author(s):

Zongyuan Chen ◽

William R. Abrams ◽

Eran Geva ◽

Claudia J. de Dood ◽

Jesús M. González ◽

...

Keyword(s):

Nucleic Acid ◽

Microfluidic Device ◽

Modular Design ◽

Simultaneous Detection ◽

Lateral Flow ◽

Nucleic Acid Amplification ◽

Antibody Detection ◽

Confirmatory Test ◽

Nucleic Acid Detection ◽

Sample Processing

A prototype dual-path microfluidic device (Rheonix CARD) capable of performing simultaneously screening (antigen or antibody) and confirmatory (nucleic acid) detection of pathogens is described. The device fully integrates sample processing, antigen or antibody detection, and nucleic acid amplification and detection, demonstrating rapid and inexpensive “sample-to-result” diagnosis with performance comparable to benchtop analysis. For the chip design, a modular approach was followed allowing the optimization of individual steps in the sample processing process. This modular design provides great versatility accommodating different disease targets independently of the production method. In the detection module, a lateral flow (LF) protocol utilizing upconverting phosphor (UCP) reporters was employed. The nucleic acid (NA) module incorporates a generic microtube containing dry reagents. Lateral flow strips and PCR primers determine the target or disease that is diagnosed. Diagnosis of HIV infection was used as a model to investigate the simultaneous detection of both human antibodies against the virus and viral RNA. The serological result is available in less than 30 min, and the confirmation by RNA amplification takes another 60 min. This approach combines a core serological portable diagnostic with a nucleic acid-based confirmatory test.

Download Full-text