Graphene oxide and self-avoiding molecular recognition systems-assisted recombinase polymerase amplification coupled with lateral flow bioassay for nucleic acid detection

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 Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV−2

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.680728 ◽

2021 ◽

Vol 11 ◽

Author(s):

Pei Wang ◽

Chao Ma ◽

Xue Zhang ◽

Lizhan Chen ◽

Longyu Yi ◽

...

Keyword(s):

Nucleic Acid ◽

Rapid Detection ◽

Simple Procedure ◽

Quantitative Polymerase Chain Reaction ◽

Recombinase Polymerase Amplification ◽

Clinical Samples ◽

Nucleic Acid Detection ◽

High Concentration ◽

Sample Processing ◽

Low Efficiency

The pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to more than 117 million reported cases and 2.6 million deaths. Accurate diagnosis technologies are vital for controlling this pandemic. Reverse transcription (RT)-based nucleic acid detection assays have been developed, but the strict sample processing requirement of RT has posed obstacles on wider applications. This study established a ligation and recombinase polymerase amplification (L/RPA) combined assay for rapid detection of SARS-CoV−2 on genes N and ORF1ab targeting the specific biomarkers recommended by the China CDC. Ligase-based strategies usually have a low-efficiency problem on RNA templates. This study has addressed this problem by using a high concentration of the T4 DNA ligase and exploiting the high sensitivity of RPA. Through selection of the ligation probes and optimization of the RPA primers, the assay achieved a satisfactory sensitivity of 101 viral RNA copies per reaction, which was comparable to RT-quantitative polymerase chain reaction (RT-qPCR) and other nucleic acid detection assays for SARS-CoV−2. The assay could be finished in less than 30 min with a simple procedure, in which the requirement for sophisticated thermocycling equipment had been avoided. In addition, it avoided the RT procedure and could potentially ease the requirement for sample processing. Once validated with clinical samples, the L/RPA assay would increase the practical testing availability of SARS-CoV-2. Moreover, the principle of L/RPA has an application potential to the identification of concerned mutations of the virus.

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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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Electron Acceptors Co-Regulated Self-Powered Photoelectrochemical Strategy and Its Application for Circulating Tumor Nucleic Acid Detection Coupled with Recombinase Polymerase Amplification

Analytical Chemistry ◽

10.1021/acs.analchem.0c01893 ◽

2020 ◽

Vol 92 (17) ◽

pp. 11771-11778 ◽

Cited By ~ 2

Author(s):

Xu Hun ◽

Yuchan Meng

Keyword(s):

Nucleic Acid ◽

Electron Acceptors ◽

Recombinase Polymerase Amplification ◽

Nucleic Acid Detection ◽

Self Powered

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Optical Graphene-Based Biosensor for Nucleic Acid Detection; Influence of Graphene Functionalization and Ionic Strength

International Journal of Molecular Sciences ◽

10.3390/ijms19103230 ◽

2018 ◽

Vol 19 (10) ◽

pp. 3230 ◽

Cited By ~ 6

Author(s):

Diana Becheru ◽

George Vlăsceanu ◽

Adela Banciu ◽

Eugeniu Vasile ◽

Mariana Ioniţă ◽

...

Keyword(s):

Electron Microscopy ◽

Graphene Oxide ◽

Ionic Strength ◽

Nucleic Acid ◽

Molecular Interaction ◽

Rational Design ◽

Nucleic Acid Detection ◽

Fluorescence Signal ◽

Main Challenge ◽

Graphene Functionalization

A main challenge for optical graphene-based biosensors detecting nucleic acid is the selection of key parameters e.g. graphenic chemical structure, nanomaterial dispersion, ionic strength, and appropriate molecular interaction mechanisms. Herein we study interactions between a fluorescein-labelled DNA (FAM-DNA) probe and target single-stranded complementary DNA (cDNA) on three graphenic species, aiming to determine the most suitable platform for nucleic acid detection. Graphene oxide (GO), carboxyl graphene (GO-COOH) and reduced graphene oxide functionalized with PEGylated amino groups (rGO-PEG-NH2, PEG (polyethylene glycol)) were dispersed and characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The influence of ionic strength on molecular interaction with DNA was examined by fluorescence resonance energy transfer (FRET) comparing fluorescence intensity and anisotropy. Results indicated an effect of graphene functionalization, dispersion and concentration-dependent quenching, with GO and GO-COOH having the highest quenching abilities for FAM-DNA. Furthermore, GO and GO-COOH quenching was accentuated by the addition of either MgCl2 or MgSO4 cations. At 10 mM MgCl2 or MgSO4, the cDNA induced a decrease in fluorescence signal that was 2.7-fold for GO, 3.4-fold for GO-COOH and 4.1-fold for rGO-PEG-NH2. Best results, allowing accurate target detection, were observed when selecting rGO-PEG-NH2, MgCl2 and fluorescence anisotropy as an advantageous combination suitable for nucleic acid detection and further rational design biosensor development.

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