scholarly journals Rapidemic, a versatile and label-free DNAzyme-based platform for visual nucleic acid detection

2020 ◽  
Author(s):  
Marijn van den Brink ◽  
Sebastian T. Tandar ◽  
Tim A. P. van den Akker ◽  
Sinisha Jovikj ◽  
Violette Defourt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Detection Method ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Nucleic Acid Detection ◽  
Label Free ◽  
Strand Displacement Amplification ◽  
Sequence Detection ◽  
Accessible Point

AbstractIn the last three decades, there have been recurring outbreaks of infectious diseases, brought to light with the recent outbreak of coronavirus disease 2019 (COVID-19). Attempts to effectively contain the spread of infectious diseases have been hampered by the lack of rapidly adaptable, accurate, and accessible point-of-care diagnostic testing. In this study, we present a novel design of a label-free DNAzyme-based detection method called Rapidemic. This assay combines recombinase polymerase amplification (RPA) with linear strand-displacement amplification (LSDA) and guanine-quadruplex (GQ) DNAzyme-catalysed colour-changing reaction. The colorimetry basis of the signal readout omits the need for extensive instrumentation. Moreover, the primer-based sequence detection of RPA gives Rapidemic a potential to be rapidly adapted to target a new sequence. As a proof of concept, we developed the assay to detect isolated genomic DNA of Saccharomyces cerevisiae. The use of low-pH buffers and the optimization of the dilution rates from each preceding reaction to the next showed to be successful strategies to enable visible detection with this method. These findings demonstrate for the first time that a label-free DNAzyme-based detection method can be coupled to RPA and LSDA for nucleic acid detection.

scholarly journals Highly Specific and Sensitive Detection of Yersinia pestis by Portable Cas12a-UPTLFA Platform

2021 ◽  
Vol 12 ◽  
Author(s):  
Yang You ◽  
Pingping Zhang ◽  
Gengshan Wu ◽  
Yafang Tan ◽  
Yong Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Yersinia Pestis ◽  
Rapid Detection ◽  
Detection Method ◽  
Detection System ◽  
Point Of Care ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Great Promise ◽  
High Background

The recent discovery of collateral cleavage activity of class-II clustered regularly interspaced short palindromic repeats–CRISPR-associated protein (CRISPR-Cas) makes CRISPR-based diagnosis a potential high-accuracy nucleic acid detection method. Colloidal gold-based lateral flow immunochromatographic assay (LFA), which has been combined with CRISPR/Cas-based nucleic detection, usually associates with drawbacks of relative high background and the subjectivity in naked-eye read-out of the results. Here, we developed a novel system composed of Cas12a-based nucleic acid detection and up-converting phosphor technology (UPT)-based LFA (UPT–LFA), termed Cas12a-UPTLFA. We further demonstrated the utility of this platform in highly sensitive and specific detection of Yersinia pestis, the causative agent of the deadly plague. Due to high infectivity and mortality, as well as the potential to be misused as bioterrorism agent, a culture-free, ultrasensitive, specific, and rapid detection method for Y. pestis has long been desired. By incorporating isothermal recombinase polymerase amplification, the Cas12a-UPTLFA we established can successfully detect genomic DNA of Y. pestis as low as 3 attomolar (aM) and exhibited high sensitivity (93.75%) and specificity (90.63%) for detection of spiked blood samples with a detection limit of 102 colony-forming unit per 100 μl of mouse blood. With a portable biosensor, Cas12a-UPTLFA assay can be operated easily by non-professional personnel. Taken together, we have developed a novel Cas12a-UPTLFA platform for rapid detection of Y. pestis with high sensitivity and specificity, which is portable, not expensive, and easy to operate as a point-of-care method. This detection system can easily be extended to detect other pathogens and holds great promise for on-site detection of emerging infectious pathogens.

scholarly journals Quality Management for Point-Of-Care Testing of Pathogen Nucleic Acids: Chinese Expert Consensus

2021 ◽  
Vol 11 ◽  
Author(s):  
Xi Mo ◽  
Xueliang Wang ◽  
Zhaoqin Zhu ◽  
Yuetian Yu ◽  
Dong Chang ◽  
...  
Keyword(s):  
Public Health ◽  
Nucleic Acid ◽  
Quality Management ◽  
Clinical Laboratory ◽  
Point Of Care ◽  
Conventional Polymerase Chain Reaction ◽  
Laboratory Medicine ◽  
Expert Consensus ◽  
Nucleic Acid Detection ◽  
Point Of Care Testing

COVID-19 continues to circulate globally in 2021, while under the precise policy implementation of China’s public health system, the epidemic was quickly controlled, and society and the economy have recovered. During the pandemic response, nucleic acid detection of SARS-CoV-2 has played an indispensable role in the first line of defence. In the cases of emergency operations or patients presenting at fever clinics, nucleic acid detection is required to be performed and reported quickly. Therefore, nucleic acid point-of-care testing (POCT) technology for SARS-CoV-2 identification has emerged, and has been widely carried out at all levels of medical institutions. SARS-CoV-2 POCT has served as a complementary test to conventional polymerase chain reaction (PCR) batch tests, thus forming an experimental diagnosis platform that not only guarantees medical safety but also improves quality services. However, in view of the complexity of molecular diagnosis and the biosafety requirements involved, pathogen nucleic acid POCT is different from traditional blood-based physical and chemical index detection. No guidelines currently exist for POCT quality management, and there have been inconsistencies documented in practical operation. Therefore, Shanghai Society of Molecular Diagnostics, Shanghai Society of Laboratory Medicine, Clinical Microbiology Division of Shanghai Society of Microbiology and Shanghai Center for Clinical Laboratory have cooperated with experts in laboratory medicine to generate the present expert consensus. Based on the current spectrum of major infectious diseases in China, the whole-process operation management of pathogen POCT, including its application scenarios, biosafety management, personnel qualification, performance verification, quality control, and result reporting, are described here. This expert consensus will aid in promoting the rational application and robust development of this technology in public health defence and hospital infection management.

scholarly journals Virus detection via programmable Type III-A CRISPR-Cas systems

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sagar Sridhara ◽  
Hemant N. Goswami ◽  
Charlisa Whyms ◽  
Jonathan H. Dennis ◽  
Hong Li
Keyword(s):  
Nucleic Acid ◽  
Detection Method ◽  
Virus Detection ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Immune Effector ◽  
Type Iii ◽  
System A ◽  
Acid Cleavage

AbstractAmong the currently available virus detection assays, those based on the programmable CRISPR-Cas enzymes have the advantage of rapid reporting and high sensitivity without the requirement of thermocyclers. Type III-A CRISPR-Cas system is a multi-component and multipronged immune effector, activated by viral RNA that previously has not been repurposed for disease detection owing in part to the complex enzyme reconstitution process and functionality. Here, we describe the construction and application of a virus detection method, based on an in vivo-reconstituted Type III-A CRISPR-Cas system. This system harnesses both RNA- and transcription-activated dual nucleic acid cleavage activities as well as internal signal amplification that allow virus detection with high sensitivity and at multiple settings. We demonstrate the use of the Type III-A system-based method in detection of SARS-CoV-2 that reached 2000 copies/μl sensitivity in amplification-free and 60 copies/μl sensitivity via isothermal amplification within 30 min and diagnosed SARS-CoV-2-infected patients in both settings. The high sensitivity, flexible reaction conditions, and the small molecular-driven amplification make the Type III-A system a potentially unique nucleic acid detection method with broad applications.

scholarly journals A Chemical-Enhanced System for CRISPR-Based Nucleic Acid Detection

2021 ◽  
Author(s):  
Zihan Li ◽  
Wenchang Zhao ◽  
Shixin Ma ◽  
Zexu Li ◽  
Yingjia Yao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Detection System ◽  
Point Of Care ◽  
Detection Sensitivity ◽  
Nucleic Acid Detection ◽  
Chemical Additives ◽  
Lateral Flow Strip ◽  
Viral Pathogens ◽  
Detection Systems ◽  
System Robustness

The CRISPR-based nucleic acid detection systems such as SHERLOCK, DETECTR and HOLMES have shown great potential for point-of-care testing of viral pathogens, especially in the context of COVID-19 pandemic. Here we optimize several key parameters of reaction chemistry and develop a Chemical Enhanced CRISPR Detection system for nucleic acid (termed CECRID). For the Cas12a/Cas13a-based signal detection phase, we determine buffer conditions and substrate range for optimal detection performance. By comparing several chemical additives, we find that addition of L-proline can secure or enhance Cas12a/Cas13a detection capability. For isothermal amplification phase with typical LAMP and RPA methods, inclusion of L-proline can also enhance specific target amplification as determined by CRISPR detection. Using SARS-CoV-2 pseudovirus, we demonstrate CECRID has enhanced detection sensitivity over chemical additive-null method with either fluorescence or lateral flow strip readout. Thus, CECRID provides an improved detection power and system robustness towards practical application of CRISPR-based diagnostics.

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 High-risk HPV nucleic acid detection kit–the careHPV test –a new detection method for screening

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Hong Ying ◽  
Fang Jing ◽  
Zhao Fanghui ◽  
Qiao Youlin ◽  
Hu Yali
Keyword(s):  
Nucleic Acid ◽  
High Risk ◽  
Detection Method ◽  
Nucleic Acid Detection ◽  
High Risk Hpv

scholarly journals Glycan heterogeneity on gold nanoparticles increases lectin discrimination capacity in label-free multiplexed bioassays

The Analyst ◽  
2016 ◽  
Vol 141 (14) ◽  
pp. 4305-4312 ◽  
Author(s):  
Lucienne Otten ◽  
Denise Vlachou ◽  
Sarah-Jane Richards ◽  
Matthew I. Gibson
Keyword(s):  
Gold Nanoparticles ◽  
Infectious Diseases ◽  
Point Of Care ◽  
Biological Warfare ◽  
Drug Resistant ◽  
Label Free ◽  
Biological Warfare Agents ◽  
Warfare Agents ◽  
Glycan Heterogeneity ◽  
Analytical Tools

The development of new analytical tools as point-of-care biosensors is crucial to combat the spread of infectious diseases, especially in the context of drug-resistant organisms, or to detect biological warfare agents.

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