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1.
Journal of analysis and testing ; : 1-9, 2022.
Article in English | EuropePMC | ID: covidwho-1710917

ABSTRACT

The COVID-19 pandemic has brought great challenges to traditional nucleic acid detection technology. Thus, it is urgent to develop a more simple and efficient nucleic acid detection technology. CRISPR-Cas12 has signal amplification ability, high sensitivity and high nucleic acid recognition specificity, so it is considered as a nucleic acid detection tool with broad development prospects and high application value. This review paper discusses recent advances in CRISPR-Cas12-based nucleic acid detection, with an emphasis on the new research methods and means to improve the nucleic acid detection capability of CRISPR-Cas12. Strategies for improving sensitivity, optimization of integrated detection, development of simplified detection mode and improvement of quantitative detection capabilities are included. Finally, the future development of CRISPR-Cas12-based nucleic acids detection is prospected.

2.
Nat Commun ; 13(1): 968, 2022 02 18.
Article in English | MEDLINE | ID: covidwho-1705624

ABSTRACT

DNA/RNA-gold nanoparticle (DNA/RNA-AuNP) nanoprobes have been widely employed for nanobiotechnology applications. Here, we discover that both thiolated and non-thiolated DNA/RNA can be efficiently attached to AuNPs to achieve high-stable spherical nucleic acid (SNA) within minutes under a domestic microwave (MW)-assisted heating-dry circumstance. Further studies show that for non-thiolated DNA/RNA the conjugation is poly (T/U) tag dependent. Spectroscopy, test strip hybridization, and loading counting experiments indicate that low-affinity poly (T/U) tag mediates the formation of a standing-up conformation, which is distributed in the outer layer of SNA structure. In further application studies, CRISPR/Cas9-sgRNA (136 bp), SARS-CoV-2 RNA fragment (1278 bp), and rolling circle amplification (RCA) DNA products (over 1000 bp) can be successfully attached on AuNPs, which overcomes the routine methods in long-chain nucleic acid-AuNP conjugation, exhibiting great promise in biosensing and nucleic acids delivery applications. Current heating-dry strategy has improved traditional DNA/RNA-AuNP conjugation methods in simplicity, rapidity, cost, and universality.


Subject(s)
Biosensing Techniques/methods , Gold/chemistry , Metal Nanoparticles/chemistry , Biotechnology/methods , COVID-19/diagnosis , COVID-19/virology , COVID-19 Nucleic Acid Testing/methods , DNA/chemistry , Heating/methods , Humans , Limit of Detection , Microwaves , Nanomedicine/methods , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/isolation & purification , SARS-CoV-2/genetics
3.
Biosens Bioelectron ; 196: 113701, 2022 Jan 15.
Article in English | MEDLINE | ID: covidwho-1638371

ABSTRACT

Although CRISPR-Cas12a and CRISPR-Cas13a systems work individually effective on gene detection, their multiplex detection capability is limited due to the lack of specific probe cleavage mechanism. Herein we present a high-efficient dual-gene diagnostic technique based on the orthogonal DNA/RNA collateral cleavage mechanism of Cas12a/Cas13a system. In this design, dual-gene amplified products from the multiplex recombinase polymerase amplification (RPA) were simultaneously detected by Cas12a and Cas13a assay in a single tube. The resulting orthogonal DNA/RNA collateral cleavage can specifically illuminate two spectral differentiated DNA and RNA probes, respectively. By integrating with the smartphone-based fluorescence readout, a portable detection platform is achieved. As a proof-of-concept, reliable dual-gene detection of SARS-CoV-2 and African Swine fever virus (ASFV) were demonstrated, exhibiting 100% sensitivity and specificity for clinical samples analysis (32 swab specimens for SARS-CoV-2 and 35 ASFV suspected swine blood samples). This developed portable dual-gene detection platform can provide accurate point-of-care screening of infectious diseases in resources-limited settings.


Subject(s)
African Swine Fever Virus , Biosensing Techniques , COVID-19 , Animals , CRISPR-Cas Systems/genetics , Humans , SARS-CoV-2 , Swine
4.
Methods ; 2021 Nov 20.
Article in English | MEDLINE | ID: covidwho-1521610

ABSTRACT

The construction of a rapid, simple, and specific nucleic acid detection platform is of great significance to the control of the large-scale spread of infectious diseases. We have recently established a magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system (termed M-CDC), which effectively integrates the advantages of CRISPR/Cas12a, magnetic beads-based separation, and AuNP bioprobe to provide a simple and specific biosensing platform for nucleic acid assay. The M-CDC method is compatible with point-of-care testing and enables the detection of nucleic acid samples in less than an hour without relying on expensive and complex instruments. In this paper, step-by-step instructions for M-CDC assay, including recombinase polymerase amplification (RPA)/reverse transcription-polymerase chain reaction (RT-RPA) of DNA or RNA, Cas12a-mediated target recognition and cleavage, and subsequent magnetic beads-mediated colorimetric readouts are provided. In addition, the protocol for the expression and purification of Lachnospiraceae bacterium-Cas12a (LbCas12a) protein, the design and synthesis of high-efficient crRNA, and the preparation of AuNP bioprobe are also offered.

5.
J Transl Int Med ; 9(2): 131-142, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1332092

ABSTRACT

BACKGROUND AND OBJECTIVES: The majority of coronavirus disease 2019 (COVID-19) cases are nonsevere, but severe cases have high mortality and need early detection and treatment. We aimed to develop a nomogram to predict the disease progression of nonsevere COVID-19 based on simple data that can be easily obtained even in primary medical institutions. METHODS: In this retrospective, multicenter cohort study, we extracted data from initial simple medical evaluations of 495 COVID-19 patients randomized (2:1) into a development cohort and a validation cohort. The progression of nonsevere COVID-19 was recorded as the primary outcome. We built a nomogram with the development cohort and tested its performance in the validation cohort. RESULTS: The nomogram was developed with the nine factors included in the final model. The area under the curve (AUC) of the nomogram scoring system for predicting the progression of nonsevere COVID-19 into severe COVID-19 was 0.875 and 0.821 in the development cohort and validation cohort, respectively. The nomogram achieved a good concordance index for predicting the progression of nonsevere COVID-19 cases in the development and validation cohorts (concordance index of 0.875 in the development cohort and 0.821 in the validation cohort) and had well-fitted calibration curves showing good agreement between the estimates and the actual endpoint events. CONCLUSIONS: The proposed nomogram built with a simplified index might help to predict the progression of nonsevere COVID-19; thus, COVID-19 with a high risk of disease progression could be identified in time, allowing an appropriate therapeutic choice according to the potential disease severity.

6.
Nano Lett ; 21(11): 4643-4653, 2021 06 09.
Article in English | MEDLINE | ID: covidwho-1303734

ABSTRACT

DNA quantification is important for biomedical research, but the routinely used techniques rely on nucleic acid amplification which have inherent issues like cross-contamination risk and quantification bias. Here, we report a CRISPR-Cas12a-based molecular diagnostic technique for amplification-free and absolute quantification of DNA at the single-molecule level. To achieve this, we first screened out the optimal reaction parameters for high-efficient Cas12a assay, yielding over 50-fold improvement in sensitivity compared with the reported Cas12a assays. We further leveraged the microdroplet-enabled confinement effect to perform an ultralocalized droplet Cas12a assay, obtaining excellent specificity and single-molecule sensitivity. Moreover, we demonstrated its versatility and quantification capability by direct counting of diverse virus's DNAs (African swine fever virus, Epstein-Barr virus, and Hepatitis B virus) from clinical serum samples with a wide range of viral titers. Given the flexible programmability of crRNA, we envision this amplification-free technique as a versatile and quantitative platform for molecular diagnosis.


Subject(s)
African Swine Fever Virus , Epstein-Barr Virus Infections , African Swine Fever Virus/genetics , Animals , CRISPR-Cas Systems , DNA/genetics , Herpesvirus 4, Human , Swine
7.
ACS Sens ; 6(3): 1086-1093, 2021 03 26.
Article in English | MEDLINE | ID: covidwho-1120724

ABSTRACT

The outbreak of COVID-19 caused a worldwide public health crisis. Large-scale population screening is an effective means to control the spread of COVID-19. Reverse transcription-polymerase chain reaction (RT-qPCR) and serology assays are the most available techniques for SARS-CoV-2 detection; however, they suffer from either less sensitivity and accuracy or low instrument accessibility for screening. To balance the sensitivity, specificity, and test availability, here, we developed enhanced colorimetry, which is termed as a magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system (M-CDC), for SARS-CoV-2 detection. By this method, SARS-CoV-2 RNA from synthetic sequences and cultured viruses can be detected by the naked eye based on gold nanoparticle (AuNP) probes, with a detection limit of 50 RNA copies per reaction. With CRISPR/Cas12a-assisted detection, SARS-CoV-2 can be specifically distinguished from other closely related viruses. M-CDC was further used to analyze 41 clinical samples, whose performance was 95.12%, consistent with that of an approved Clinical RT-qPCR Diagnosis kit. The developed M-CDC method is not dependent on sophisticated instruments, which makes it potentially valuable to be applied for SARS-CoV-2 screening under poor conditions.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , RNA, Viral/analysis , SARS-CoV-2/genetics , Bacterial Proteins , CRISPR-Associated Proteins , CRISPR-Cas Systems , Cell Line, Tumor , Colorimetry , DNA/chemistry , DNA Probes , Endodeoxyribonucleases , Gold/chemistry , Humans , Metal Nanoparticles/chemistry
8.
Biotechnol Bioeng ; 118(5): 2053-2066, 2021 05.
Article in English | MEDLINE | ID: covidwho-1092501

ABSTRACT

Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol contamination, especially when an end-point detection mode is applied. Here, we proposed a solution by designing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 eraser strategy for eliminating potential contamination amplification. The CRISPR/Cas9 engineered eraser is firstly adopted into artpcr reverse-transcription PCR (RT-PCR) system to achieve contamination-free RNA detection. Subsequently, we extended this CRISPR/Cas9 eraser to the PCR system. We engineered conventional PCR primers to enable the amplified products to contain an implanted NGG (protospacer adjacent motif, PAM) site, which is used as a code for specific CRISPR/Cas9 recognition. Pre-incubation of Cas9/sgRNA with PCR mix leads to a selective cleavage of contamination amplicons, thus only the template DNA is amplified. The developed CRISPR/Cas9 eraser, adopted by both RT-PCR and PCR systems, showed high-fidelity detection of SARS-CoV-2 and African swine fever virus with a convenient strip test.


Subject(s)
CRISPR-Cas Systems , Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , African Swine Fever Virus/isolation & purification , Animals , Humans , RNA, Guide , RNA, Viral/isolation & purification , SARS-CoV-2/isolation & purification , Swine
9.
Angewandte Chemie ; 133(10):5367-5375, 2021.
Article in English | ProQuest Central | ID: covidwho-1092137

ABSTRACT

Few methods for the detection of SARS‐CoV‐2 currently have the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT‐qPCR method. Developed here is a CRISPR/Cas9‐mediated triple‐line lateral flow assay (TL‐LFA) combined with multiplex reverse transcription‐recombinase polymerase amplification (RT‐RPA) for rapid and simultaneous dual‐gene detection of SARS‐CoV‐2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (Orf1ab) genes from cell‐cultured SARS‐CoV‐2 and SARS‐CoV‐2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 μL). Furthermore, dual‐gene analysis of 64 nasopharyngeal swab samples showed 100 % negative predictive agreement and 97.14 % positive predictive agreement. This platform will provide a more accurate and convenient pathway for diagnosis of COVID‐19 or other infectious diseases in low‐resource regions.

10.
ACS Nano ; 15(1): 1167-1178, 2021 01 26.
Article in English | MEDLINE | ID: covidwho-1014984

ABSTRACT

Existing methods for RNA diagnostics, such as reverse transcription PCR (RT-PCR), mainly rely on nucleic acid amplification (NAA) and RT processes, which are known to introduce substantial issues, including amplification bias, cross-contamination, and sample loss. To address these problems, we introduce a confinement effect-inspired Cas13a assay for single-molecule RNA diagnostics, eliminating the need for NAA and RT. This assay involves confining the RNA-triggered Cas13a catalysis system in cell-like-sized reactors to enhance local concentrations of target and reporter simultaneously, via droplet microfluidics. It achieves >10 000-fold enhancement in sensitivity when compared to the bulk Cas13a assay and enables absolute digital single-molecule RNA quantitation. We experimentally demonstrate its broad applicability for precisely counting microRNAs, 16S rRNAs, and SARS-CoV-2 RNA from synthetic sequences to clinical samples with excellent accuracy. Notably, this direct RNA diagnostic technology enables detecting a wide range of RNA molecules at the single-molecule level. Moreover, its simplicity, universality, and excellent quantification capability might render it to be a dominant rival to RT-qPCR.


Subject(s)
CRISPR-Cas Systems , Microfluidics , RNA/analysis , Cell Line, Tumor , Enterococcus faecalis , Escherichia coli , Humans , Klebsiella pneumoniae , MCF-7 Cells , MicroRNAs/analysis , Pseudomonas aeruginosa , RNA, Ribosomal, 16S/analysis , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Staphylococcus aureus
11.
Angew Chem Int Ed Engl ; 60(10): 5307-5315, 2021 03 01.
Article in English | MEDLINE | ID: covidwho-970071

ABSTRACT

Few methods for the detection of SARS-CoV-2 currently have the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Developed here is a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (Orf1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 µL). Furthermore, dual-gene analysis of 64 nasopharyngeal swab samples showed 100 % negative predictive agreement and 97.14 % positive predictive agreement. This platform will provide a more accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in low-resource regions.


Subject(s)
COVID-19/diagnosis , CRISPR-Cas Systems , Genes, Viral , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , COVID-19/virology , Humans , Nasopharynx/virology , RNA, Viral , SARS-CoV-2/genetics , Sensitivity and Specificity
12.
Med Sci Monit ; 26: e925364, 2020 Aug 06.
Article in English | MEDLINE | ID: covidwho-696161

ABSTRACT

Traced back to December 2019, an unexpected outbreak of a highly contagious new coronavirus pneumonia (COVID-19) has rapidly swept around China and the globe. There have now been an estimated 2 580 000 infections and more than 170 000 fatal cases around the world. The World Health Organization (WHO) estimated that approximately 14% of infections developed into severe disease, 5% were critically ill, and the mortality rate of critically ill patients is reported to be over 50%. The shortage of specific anti-viral treatment and vaccines remains a huge challenge. In COVID-19, refractory hypoxemia is common among the critically ill with acute respiratory distress syndrome (ARDS) despite invasive mechanical ventilation, and is further complicated by respiratory and circulatory failure. This difficult situation calls for the use of extracorporeal membrane oxygenation (ECMO) for assisting respiration and circulation if necessary. This article reviews the pertinent clinical literature, technical guidance, and expert recommendations on use of ECMO in critically ill cases of COVID-19. Here, we present basic knowledge and opinions about COVID-19 and ECMO, review the evidence on ECMO use in Middle East Respiratory Syndrome (MERS) and H1N1 influenza, share the technical guidance and recommendations on use of ECMO in COVID-19, summarize the current use of ECMO against COVID-19 in China, and discuss the issues in use of ECMO in COVID-19.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/therapy , Critical Illness , Extracorporeal Membrane Oxygenation , Pneumonia, Viral/therapy , Pneumonia/therapy , Respiratory Distress Syndrome/therapy , COVID-19 , Humans , Pandemics , Practice Guidelines as Topic , SARS-CoV-2
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