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 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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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.

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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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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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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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Isothermal Recombinase Polymerase Amplification-lateral Flow Detection of SARS-CoV-2, the Etiological Agent of COVID-19

10.21203/rs.3.rs-78408/v2 ◽

2020 ◽

Author(s):

Thomas R Shelite ◽

Ashanti C Uscanga-Palomeque ◽

Alejandro Castellanos ◽

Peter C Melby ◽

Bruno L Travi

Keyword(s):

Rapid Detection ◽

Limit Of Detection ◽

Point Of Care ◽

The United States ◽

Lateral Flow ◽

Recombinase Polymerase Amplification ◽

N Gene ◽

Clinical Samples ◽

Reference Test ◽

Delay In Diagnosis

Abstract The rapid detection of novel pathogens necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. In December of 2019, novel coronavirus, SARS-CoV-2 (2019-nCoV), was isolated from a cluster of pneumonia patients in the Chinese city of Wuhan. The virus rapidly spread throughout the world and the first fatal cases of COVID-19 in the United States occurred in late February. The lack of testing and delay in diagnosis has facilitated the spread of this novel virus. Development of point-of-care diagnostic assays that can be performed in rural or decentralized health care centers to expand testing capacity is needed. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of SARS-CoV-2. The RPA-LF detected SARS-CoV-2 with a limit of detection of 35.4 viral nucleocapsid (N) gene copies/µL. Additionally, the RPA-LF was able to detect 0.25-2.5 copies/µL of SARS-CoV-2 N gene containing plasmid. We evaluated 37 clinical samples using CDC’s N3, N1 and N2 RT-real-time PCR assays for SARS-CoV-2 as reference test. We found a 100% concordance between RPA-LF and RT-qPCR reference test as determined by 18/18 positive and 19/19 negative samples. All positive samples had Ct values between 19-37 by RT-qPCR. The RPA-LF primers and probe did not cross react with other relevant betacoronaviruses such as SARS and MERS. This is the first isothermal amplification test paired with lateral flow developed for qualitative detection of COVID-19 allowing rapid viral detection and with prospective applicability in resource limited and decentralized laboratories.

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Isothermal Recombinase Polymerase Amplification-lateral Flow Detection of SARS-CoV-2, the Etiological Agent of COVID-19

10.21203/rs.3.rs-78408/v1 ◽

2020 ◽

Author(s):

Thomas R Shelite ◽

Ashanti C Uscanga-Palomeque ◽

Alejandro Castellanos ◽

Peter C Melby ◽

Bruno L Travi

Keyword(s):

Rapid Detection ◽

Limit Of Detection ◽

Point Of Care ◽

The United States ◽

Lateral Flow ◽

Recombinase Polymerase Amplification ◽

N Gene ◽

Clinical Samples ◽

Reference Test ◽

Delay In Diagnosis

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Comparative Study of Gold and Carbon Nanoparticles in Nucleic Acid Lateral Flow Assay

Nanomaterials ◽

10.3390/nano11030741 ◽

2021 ◽

Vol 11 (3) ◽

pp. 741

Author(s):

Juan Carlos Porras ◽

Mireia Bernuz ◽

Jennifer Marfa ◽

Arnau Pallares-Rusiñol ◽

Mercè Martí ◽

...

Keyword(s):

Nucleic Acid ◽

Comparative Study ◽

Carbon Nanoparticles ◽

Limit Of Detection ◽

Early Stage ◽

High Sensitivity ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Complex Samples ◽

Increased Sensitivity

A lateral flow assay (LFA) is a paper-based, point-of-need test designed to detect a specific analyte in complex samples in low-resource settings. Although LFA has been successfully used in different applications, its use is still limited when high sensitivity is required, especially in the diagnosis of an early-stage condition. The limit of detection (LOD) is clearly related to the signal-generating system used to achieve the visual readout, in many cases involving nanoparticles coupled to a biomolecule, which, when combined, provides sensitivity and specificity, respectively. While colloidal gold is currently the most-used label, other detection systems are being developed. Carbon nanoparticles (CNPs) demonstrate outstanding features to improve the sensitivity of this technology by producing an increased contrast in the paper background. Based on the necessity of sensitivity improvement, the aim of this work is a comparative study, in terms of analytical performance, between commercial streptavidin gold nanoparticles (streptAv-AuNPs) and avidin carbon nanoparticles (Av-CNPs) in a nucleic acid lateral flow assay. The visual LOD of the method was calculated by serial dilution of the DNA template, ranging from 0.0 to 7 pg μL−1/1.5 × 104 CFU mL−1). The LFA achieved visual detection of as low as 2.2 × 10−2 pg μL−1 using Av-CNPs and 8.4 × 10−2 pg μL−1 using streptAv-AuNPs. These LODs could be obtained without the assistance of any instrumentation. The results demonstrate that CNPs showed an increased sensitivity, achieving the nanomolar range even by visual inspection. Furthermore, CNPs are the cheapest labels, and the suspensions are very stable and easy to modify.

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